Masson P

General

Full name : Masson Patrick

First name : Patrick

Mail : Neuropharmacology Laboratory, Kazan Federal University, Kazan, Russia

Zip Code : 38702

City : Grenoble

Country : France

Email : pym.masson@free.fr

Phone :

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References (231)

Title : Human Plasma Butyrylcholinesterase Hydrolyzes Atropine: Kinetic and Molecular Modeling Studies - Mukhametgalieva_2024_Molecules_29_2140
Author(s) : Mukhametgalieva A , Mir SA , Shaihutdinova Z , Masson P
Ref : Molecules , 29 : , 2024
Abstract : The participation of butyrylcholinesterase (BChE) in the degradation of atropine has been recurrently addressed for more than 70 years. However, no conclusive answer has been provided for the human enzyme so far. In the present work, a steady-state kinetic analysis performed by spectrophotometry showed that highly purified human plasma BChE tetramer slowly hydrolyzes atropine at pH 7.0 and 25 degreesC. The affinity of atropine for the enzyme is weak, and the observed kinetic rates versus the atropine concentration was of the first order: the maximum atropine concentration in essays was much less than K(m). Thus, the bimolecular rate constant was found to be k(cat)/K(m) = 7.7 x 10(4) M(-1) min(-1). Rough estimates of catalytic parameters provided slow k(cat) < 40 min(-1) and high K(m) = 0.3-3.3 mM. Then, using a specific organophosphoryl agent, echothiophate, the time-dependent irreversible inhibition profiles of BChE for hydrolysis of atropine and the standard substrate butyrylthiocholine (BTC) were investigated. This established that both substrates are hydrolyzed at the same site, i.e., S198, as for all substrates of this enzyme. Lastly, molecular docking provided evidence that both atropine isomers bind to the active center of BChE. However, free energy perturbations yielded by the Bennett Acceptance Ratio method suggest that the L-atropine isomer is the most reactive enantiomer. In conclusion, the results provided evidence that plasma BChE slowly hydrolyzes atropine but should have no significant role in its metabolism under current conditions of medical use and even under administration of the highest possible doses of this antimuscarinic drug.
ESTHER : Mukhametgalieva_2024_Molecules_29_2140
PubMedSearch : Mukhametgalieva_2024_Molecules_29_2140
PubMedID: 38731631

Title : Activation\/Inhibition of Cholinesterases by Excess Substrate: Interpretation of the Phenomenological b Factor in Steady-State Rate Equation - Mukhametgalieva_2023_Int.J.Mol.Sci_24_
Author(s) : Mukhametgalieva AR , Nemtarev AV , Sykaev VV , Pashirova TN , Masson P
Ref : Int J Mol Sci , 24 : , 2023
Abstract : Cholinesterases (ChEs) display a non-michaelian behavior with positively charged substrates. In the steady-state rate equation, the b factor describes this behavior: if b > 1 there is substrate activation, if b < 1 there is substrate inhibition. The mechanistic significance of the b factor was investigated to determine whether this behavior depends on acylation, deacylation or on both steps. Kinetics of human acetyl- (AChE) and butyryl-cholinesterase (BChE) were performed under steady-state conditions and using a time-course of complete substrate hydrolysis. For the hydrolysis of short acyl(thio)esters, where acylation and deacylation are partly rate-limiting, steady-state kinetic analysis could not decide which step determines b. However, the study of the hydrolysis of an arylacylamide, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), where acetylation is rate-limiting, showed that b depends on the acylation step. The magnitude of b and opposite b values between AChE and BChE for the hydrolysis of acetyl(thio)- versus benzoyl-(thio) esters, then indicated that the productive adjustment of substrates in the active center at high concentration depends on motions of both the and the acyl-binding loops. Benzoylcholine was shown to be a poor substrate of AChE, and steady-state kinetics showed a sudden inhibition at high concentration, likely due to the non-dissociation of hydrolysis products. The poor catalytic hydrolysis of this bulky ester by AChE illustrates the importance of the fine adjustment of substrate acyl moiety in the acyl-binding pocket. Molecular modeling and QM/MM simulations should definitively provide evidence for this statement.
ESTHER : Mukhametgalieva_2023_Int.J.Mol.Sci_24_
PubMedSearch : Mukhametgalieva_2023_Int.J.Mol.Sci_24_
PubMedID: 37445649

Title : Drug and pro-drug substrates and pseudo-substrates of human butyrylcholinesterase - Masson_2023_Biochem.Pharmacol_218_115910
Author(s) : Masson P , Shaihutdinova Z , Lockridge O
Ref : Biochemical Pharmacology , 218 :115910 , 2023
Abstract : Butyrylcholinesterase (BChE) is present in plasma and numerous cells and organs. Its physiological function(s) is(are) still unclear. However, this enzyme is of pharmacological and toxicological importance. It displays a broad specificity and is capable of hydrolyzing a wide range of substrates with turnovers differing by several orders of magnitude. Nowaday, these substrates include more than two dozen carboxyl-ester drugs, numerous acetylated prodrugs, and transition state analogues of acetylcholine. In addition, BChE displays a promiscuous hydrolytic activity toward amide bonds of arylacylamides, and slowly hydrolyzes carbamyl- and phosphoryl-esters. Certain pseudo-substrates like carbamates and organophosphates are major drugs of potential medical interest. The existence of a large genetic poly-allelism, affecting the catalytic properties of BChE is at the origin of clinical complications in the use of certain drugs catabolized by BChE. The number of drugs and prodrugs hydrolyzed by BChE is expected to increase in the future. However, very few quantitative data (K(m), k(cat)) are available for most marketed drugs, and except for myorelaxants like succinylcholine and mivacurium, the impact of BChE genetic mutations on catalytic parameters has not been evaluated for most of these drugs.
ESTHER : Masson_2023_Biochem.Pharmacol_218_115910
PubMedSearch : Masson_2023_Biochem.Pharmacol_218_115910
PubMedID: 37972875

Title : Partial Reversible Inhibition of Enzymes and Its Metabolic and Pharmaco-Toxicological Implications - Masson_2023_Int.J.Mol.Sci_24_
Author(s) : Masson P , Mukhametgalieva AR
Ref : Int J Mol Sci , 24 : , 2023
Abstract : Partial reversible inhibition of enzymes, also called hyperbolic inhibition, is an uncommon mechanism of reversible inhibition, resulting from a productive enzyme-inhibitor complex. This type of inhibition can involve competitive, mixed, non-competitive and uncompetitive inhibitors. While full reversible inhibitors show linear plots for reciprocal enzyme initial velocity versus inhibitor concentration, partial inhibitors produce hyperbolic plots. Similarly, dose-response curves show residual fractional activity of enzymes at high doses. This article reviews the theory and methods of analysis and discusses the significance of this type of reversible enzyme inhibition in metabolic processes, and its implications in pharmacology and toxicology.
ESTHER : Masson_2023_Int.J.Mol.Sci_24_
PubMedSearch : Masson_2023_Int.J.Mol.Sci_24_
PubMedID: 37629158

Title : Conformational Stability and Denaturation Processes of Proteins Investigated by Electrophoresis under Extreme Conditions - Masson_2022_Molecules_27_
Author(s) : Masson P , Lushchekina S
Ref : Molecules , 27 : , 2022
Abstract : The functional structure of proteins results from marginally stable folded conformations. Reversible unfolding, irreversible denaturation, and deterioration can be caused by chemical and physical agents due to changes in the physicochemical conditions of pH, ionic strength, temperature, pressure, and electric field or due to the presence of a cosolvent that perturbs the delicate balance between stabilizing and destabilizing interactions and eventually induces chemical modifications. For most proteins, denaturation is a complex process involving transient intermediates in several reversible and eventually irreversible steps. Knowledge of protein stability and denaturation processes is mandatory for the development of enzymes as industrial catalysts, biopharmaceuticals, analytical and medical bioreagents, and safe industrial food. Electrophoresis techniques operating under extreme conditions are convenient tools for analyzing unfolding transitions, trapping transient intermediates, and gaining insight into the mechanisms of denaturation processes. Moreover, quantitative analysis of electrophoretic mobility transition curves allows the estimation of the conformational stability of proteins. These approaches include polyacrylamide gel electrophoresis and capillary zone electrophoresis under cold, heat, and hydrostatic pressure and in the presence of non-ionic denaturing agents or stabilizers such as polyols and heavy water. Lastly, after exposure to extremes of physical conditions, electrophoresis under standard conditions provides information on irreversible processes, slow conformational drifts, and slow renaturation processes. The impressive developments of enzyme technology with multiple applications in fine chemistry, biopharmaceutics, and nanomedicine prompted us to revisit the potentialities of these electrophoretic approaches. This feature review is illustrated with published and unpublished results obtained by the authors on cholinesterases and paraoxonase, two physiologically and toxicologically important enzymes.
ESTHER : Masson_2022_Molecules_27_
PubMedSearch : Masson_2022_Molecules_27_
PubMedID: 36296453

Title : alpha-tocopherol, a slow-binding inhibitor of acetylcholinesterase - Zueva_2021_Chem.Biol.Interact__109646
Author(s) : Zueva I , Lushchekina S , Shulnikova P , Lenina O , Petrov K , Molochkina E , Masson P
Ref : Chemico-Biological Interactions , :109646 , 2021
Abstract : Acetylcholinesterase (AChE) is reversibly inhibited by alpha-tocopherol (alpha-T). Steady state kinetic analysis shows that alpha-T is a mixed slow-binding inhibitor of type A of human enzyme (K(ci) = 0.49 microM; K(ui) = 1.6 microM) with a residence time of 2 min on target. Molecular dynamics (MD) simulations support this mechanism, and indicate that alpha-T first forms multiple non-specific interactions with AChE surface near the gorge entrance, then binds to the peripheral side with alkylene chain slowly sliding down the gorge, inducing no significant conformational change. alpha-T slightly modulates the progressive inhibition of AChE by the cyclic organophosphorus, cresyl saligenylphosphate, accelerating the fast pseudo-first order process of phosphorylation. A moderate accelerating effect of alpha-T on phosphorylation by paraoxon was also observed after pre-incubation of AChE in the presence of alpha-T. This accelerating effect of alpha-T on ex vivo paraoxon-induced diaphragm muscle weakness was also observed. The effect of alpha-T on AChE phosphylation was interpreted in light of molecular modeling results. From all results it is clear that alpha-T does not protect AChE against phosphylation by organophosphorus.
ESTHER : Zueva_2021_Chem.Biol.Interact__109646
PubMedSearch : Zueva_2021_Chem.Biol.Interact__109646
PubMedID: 34506764

Title : Steady-state kinetic analysis of human cholinesterases over wide concentration ranges of competing substrates - Mukhametgalieva_2021_Biochim.Biophys.Acta.Proteins.Proteom__140733
Author(s) : Mukhametgalieva AR , Lushchekina SV , Aglyamova AR , Masson P
Ref : Biochimica & Biophysica Acta Proteins Proteom , :140733 , 2021
Abstract : Substrate competition for human acetylcholinesterase (AChE) and human butyrylcholinesterase (BChE) was studies under steady-state conditions using wide range of substrate concentrations. Competing couples of substates were acetyl-(thio)esters. Phenyl acetate (PhA) was the reporter substrate and competitor were either acetylcholine (ACh) or acetylthiocholine (ATC). The common point between investigated substrates is that the acyl moiety is acetate, i.e. same deacylation rate constant for reporter and competitor substrate. Steady-state kinetics of cholinesterase-catalyzed hydrolysis of PhA in the presence of ACh or ATC revealed 3 phases of inhibition as concentration of competitor increased: a) competitive inhibition, b) partially mixed inhibition, c) partially uncompetitive inhibition for AChE and partially uncompetitive activation for BChE. This sequence reflects binding of competitor in the active centrer at low concentration and on the peripheral anionic site (PAS) at high concentration. In particular, it showed that binding of a competing ligand on PAS may affect the catalytic behavior of AChE and BChE in an opposite way, i.e. inhibition of AChE and activation of BChE, regardless the nature of the reporter substrate. For both enzymes, progress curves for hydrolysis of PhA at very low concentration (<
ESTHER : Mukhametgalieva_2021_Biochim.Biophys.Acta.Proteins.Proteom__140733
PubMedSearch : Mukhametgalieva_2021_Biochim.Biophys.Acta.Proteins.Proteom__140733
PubMedID: 34662731

Title : Protective effects of m-(tert-butyl) trifluoroacetophenone, a transition state analogue of acetylcholine, against paraoxon toxicity and memory impairments - Zueva_2021_Chem.Biol.Interact_345_109558
Author(s) : Zueva IV , Lenina OA , Kayumova RM , Petrov KA , Masson P
Ref : Chemico-Biological Interactions , 345 :109558 , 2021
Abstract : m-(Tert-butyl) trifluoroacetophenone (TFK), a slow-binding inhibitor of acetylcholinesterase (AChE), a transition state analog of acetylcholine, was investigated as a potential neuroprotectant of central and peripheral AChE against organophosphate paraoxon (POX) toxicity. Acute toxicity and pharmacological effects of TFK were investigated on mice and rats. Intraperitoneal administered TFK has low acute toxicity in mice (LD(50) = 19 mg/kg). Effects on motor function as investigated by rotarod and open field tests showed that TFK up to 5 mg/kg did not alter motor coordination and stereotypical exploration behavior of mice. Passive avoidance test showed that 1 or 5 mg/kg TFK restored memory impairment in scopolamine-induced Alzheimer's disease-like dementia in rats. Pretreatment of mice with 5 mg/kg TFK, 2-3 hrs before challenge by 2xLD(50) POX provided a modest and short protection against POX toxicity. Futhermore, analysis of POX-induced neuronal degeneration by using fluoro-jade B staining showed that TFK pretreatment, at the dose 5mg/kg before POX challenge, significantly reduced the density of apoptotic cells in hippocampus and entorhinal cortex of mice. Thus, TFK is capable of reducing POX-induced neurotoxicity.
ESTHER : Zueva_2021_Chem.Biol.Interact_345_109558
PubMedSearch : Zueva_2021_Chem.Biol.Interact_345_109558
PubMedID: 34147486

Title : Organophosphorus poisoning in animals and enzymatic antidotes - Poirier_2021_Environ.Sci.Pollut.Res.Int_28_25081
Author(s) : Poirier L , Jacquet P , Plener L , Masson P , Daude D , Chabriere E
Ref : Environ Sci Pollut Res Int , 28 :25081 , 2021
Abstract : Organophosphorus compounds (OPs) are neurotoxic molecules developed as pesticides and chemical warfare nerve agents (CWNAs). Most of them are covalent inhibitors of acetylcholinesterase (AChE), a key enzyme in nervous systems, and are therefore responsible for numerous poisonings around the world. Many animal models have been studied over the years in order to decipher the toxicity of OPs and to provide insights for therapeutic and decontamination purposes. Environmental impact on wild animal species has been analyzed to understand the consequences of OP uses in agriculture. In complement, various laboratory models, from invertebrates to aquatic organisms, rodents and primates, have been chosen to study chronic and acute toxicity as well as neurobehavioral impact, immune response, developmental disruption, and other pathological signs. Several decontamination approaches were developed to counteract the poisoning effects of OPs. Among these, enzyme-based strategies are particularly attractive as they allow efficient external decontamination without toxicity or environmental impact and may be of interest for treatment. Approaches using bioscavengers for prophylaxis, treatment, and external decontamination are emphasized and their potential is discussed in the light of toxicological observations from various animal models. The relevance of animal models, regarding their cholinergic system and the abundance of naturally protecting enzymes, is also discussed for better extrapolation of results to human.
ESTHER : Poirier_2021_Environ.Sci.Pollut.Res.Int_28_25081
PubMedSearch : Poirier_2021_Environ.Sci.Pollut.Res.Int_28_25081
PubMedID: 29959732

Title : Therapeutic nanoreactors for detoxification of xenobiotics: Concepts, challenges and biotechnological trends with special emphasis to organophosphate bioscavenging - Pashirova_2021_Chem.Biol.Interact__109577
Author(s) : Pashirova TN , Bogdanov A , Masson P
Ref : Chemico-Biological Interactions , :109577 , 2021
Abstract : The introduction of enzyme nanoreactors in medicine is relatively new. However, this technology has already been experimentally successful in cancer treatments, struggle against toxicity of reactive oxygen species in inflammatory processes, detoxification of drugs and xenobiotics, and correction of metabolic and genetic defects by using encapsulated enzymes, acting in single or cascade reactions. Biomolecules, e.g. enzymes, antibodies, reactive proteins capable of inactivating toxicants in the body are called bioscavengers. In this review, we focus on enzyme-containing nanoreactors for in vivo detoxification of organophosphorous compounds (OP) to be used for prophylaxis and post-exposure treatment of OP poisoning. A particular attention is devoted to bioscavenger-containing injectable nanoreactors operating in the bloodstream. The nanoreactor concept implements single or multiple enzymes and cofactors co-encapsulated in polymeric semi-permeable nanocontainers. Thus, the detoxification processes take place in a confined space containing highly concentrated bioscavengers. The article deals with historical and theoretical backgrounds about enzymatic detoxification of OPs in nanoreactors, nanoreactor polymeric enveloppes, realizations and advantages over other approaches using bioscavengers.
ESTHER : Pashirova_2021_Chem.Biol.Interact__109577
PubMedSearch : Pashirova_2021_Chem.Biol.Interact__109577
PubMedID: 34274336

Title : Impact of Sucrose as Osmolyte on Molecular Dynamics of Mouse Acetylcholinesterase - Lushchekina_2020_Biomolecules_10_
Author(s) : Lushchekina SV , Inidjel G , Martinez N , Masson P , Trovaslet-Leroy M , Nachon F , Koza MM , Seydel T , Peters J
Ref : Biomolecules , 10 : , 2020
Abstract : The enzyme model, mouse acetylcholinesterase, which exhibits its active site at the bottom of a narrow gorge, was investigated in the presence of different concentrations of sucrose to shed light on the protein and water dynamics in cholinesterases. The study was conducted by incoherent neutron scattering, giving access to molecular dynamics within the time scale of sub-nano to nanoseconds, in comparison with molecular dynamics simulations. With increasing sucrose concentration, we found non-linear effects, e.g., first a decrease in the dynamics at 5 wt% followed by a gain at 10 wt% sucrose. Direct comparisons with simulations permitted us to understand the following findings: at 5 wt%, sugar molecules interact with the protein surface through water molecules and damp the motions to reduce the overall protein mobility, although the motions inside the gorge are enhanced due to water depletion. When going to 10 wt% of sucrose, some water molecules at the protein surface are replaced by sugar molecules. By penetrating the protein surface, they disrupt some of the intra-protein contacts, and induce new ones, creating new pathways for correlated motions, and therefore, increasing the dynamics. This exhaustive study allowed for an explanation of the detail interactions leading to the observed non-linear behavior.
ESTHER : Lushchekina_2020_Biomolecules_10_
PubMedSearch : Lushchekina_2020_Biomolecules_10_
PubMedID: 33322722

Title : Slow-binding reversible inhibitor of acetylcholinesterase with long-lasting action for prophylaxis of organophosphate poisoning - Lenina_2020_Sci.Rep_10_16611
Author(s) : Lenina OA , Zueva IV , Zobov VV , Semenov VE , Masson P , Petrov KA
Ref : Sci Rep , 10 :16611 , 2020
Abstract : Organophosphorus (OP) compounds represent a serious health hazard worldwide. The dominant mechanism of their action results from covalent inhibition of acetylcholinesterase (AChE). Standard therapy of acute OP poisoning is partially effective. However, prophylactic administration of reversible or pseudo-irreversible AChE inhibitors before OP exposure increases the efficiency of standard therapy. The purpose of the study was to test the duration of the protective effect of a slow-binding reversible AChE inhibitor (C547) in a mouse model against acute exposure to paraoxon (POX). It was shown that the rate of inhibition of AChE by POX in vitro after pre-inhibition with C547 was several times lower than without C547. Ex vivo pre-incubation of mouse diaphragm with C547 significantly prevented the POX-induced muscle weakness. Then it was shown that pre-treatment of mice with C547 at the dose of 0.01 mg/kg significantly increased survival after poisoning by 2xLD(50) POX. The duration of the pre-treatment was effective up to 96 h, whereas currently used drug for pre-exposure treatment, pyridostigmine at a dose of 0.15 mg/kg was effective less than 24 h. Thus, long-lasting slow-binding reversible AChE inhibitors can be considered as new potential drugs to increase the duration of pre-exposure treatment of OP poisoning.
ESTHER : Lenina_2020_Sci.Rep_10_16611
PubMedSearch : Lenina_2020_Sci.Rep_10_16611
PubMedID: 33024231

Title : Steady-State Kinetics of Enzyme-Catalyzed Hydrolysis of Echothiophate, a P-S Bonded Organophosphorus as Monitored by Spectrofluorimetry - Zueva_2020_Molecules_25_
Author(s) : Zueva IV , Lushchekina SV , Daude D , Chabriere E , Masson P
Ref : Molecules , 25 : , 2020
Abstract : Enzyme-catalyzed hydrolysis of echothiophate, a P-S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with Km = 0.20 +/- 0.03 mM and kcat = 5.4 +/- 1.6 min(-1). The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (Km = 2.6 +/- 0.2 mM; kcat = 53400 min(-1)). With a kcat/Km = (2.6 +/- 1.6) x 10(7) M(-1)min(-1), GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P-S bonded OPs by thiol-free OP hydrolases.
ESTHER : Zueva_2020_Molecules_25_
PubMedSearch : Zueva_2020_Molecules_25_
PubMedID: 32192230

Title : Slow-binding inhibitors of acetylcholinesterase of medical interest - Lushchekina_2020_Neuropharmacol__108236
Author(s) : Lushchekina SV , Masson P
Ref : Neuropharmacology , :108236 , 2020
Abstract : Certain ligands slowly bind to acetylcholinesterase. As a result, there is a slow establishment of enzyme-inhibitor equilibrium characterized by a slow onset of inhibition prior reaching steady state. Three mechanisms account for slow-binding inhibition: a) slow binding rate constant k(on), b) slow ligand induced-fit following a fast binding step, c) slow conformational selection of an enzyme form. The slow equilibrium may be followed by a chemical step. This later that can be irreversible has been observed with certain alkylating agents and substrate transition state analogs. Slow-binding inhibitors present long residence times on target. This results in prolonged pharmacological or toxicological action. Through several well-known molecules (e.g. huperzine) and new examples (tocopherol, trifluoroacetophenone and a 6-methyluracil alkylammonium derivative), we show that slow-binding inhibitors of acetylcholinesterase are promising drugs for treatment of neurological diseases such as Alzheimer disease and myasthenia gravis. Moreover, they may be of interest for neuroprotection (prophylaxis) against organophosphorus poisoning.
ESTHER : Lushchekina_2020_Neuropharmacol__108236
PubMedSearch : Lushchekina_2020_Neuropharmacol__108236
PubMedID: 32712274

Title : 1-(3-Tert-Butylphenyl)-2,2,2-Trifluoroethanone as a Potent Transition-State Analogue Slow-Binding Inhibitor of Human Acetylcholinesterase: Kinetic, MD and QM\/MM Studies - Zueva_2020_Biomolecules_10_1608
Author(s) : Zueva IV , Lushchekina SV , Pottie IR , Darvesh S , Masson P
Ref : Biomolecules , 10 : , 2020
Abstract : Kinetic studies and molecular modeling of human acetylcholinesterase (AChE) inhibition by a fluorinated acetophenone derivative, 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK), were performed. Fast reversible inhibition of AChE by TFK is of competitive type with K(i) = 5.15 nM. However, steady state of inhibition is reached slowly. Kinetic analysis showed that TFK is a slow-binding inhibitor (SBI) of type B with K(i)* = 0.53 nM. Reversible binding of TFK provides a long residence time, = 20 min, on AChE. After binding, TFK acylates the active serine, forming an hemiketal. Then, disruption of hemiketal (deacylation) is slow. AChE recovers full activity in approximately 40 min. Molecular docking and MD simulations depicted the different steps. It was shown that TFK binds first to the peripheral anionic site. Then, subsequent slow induced-fit step enlarged the gorge, allowing tight adjustment into the catalytic active site. Modeling of interactions between TFK and AChE active site by QM/MM showed that the "isomerization" step of enzyme-inhibitor complex leads to a complex similar to substrate tetrahedral intermediate, a so-called "transition state analog", followed by a labile covalent intermediate. SBIs of AChE show prolonged pharmacological efficacy. Thus, this fluoroalkylketone intended for neuroimaging, could be of interest in palliative therapy of Alzheimer's disease and protection of central AChE against organophosphorus compounds.
ESTHER : Zueva_2020_Biomolecules_10_1608
PubMedSearch : Zueva_2020_Biomolecules_10_1608
PubMedID: 33260981

Title : Structural Stability of Human Butyrylcholinesterase under High Hydrostatic Pressure - Kangur_2019_Biochim.Biophys.Acta.Proteins.Proteom_1867_107
Author(s) : Kangur L , Timpmann K , Zeller D , Masson P , Peters J , Freiberg A
Ref : Biochimica & Biophysica Acta Proteins Proteom , 1867 :107 , 2019
Abstract : Human butyrylcholinesterase is a nonspecific enzyme of clinical, pharmacological and toxicological significance. Although the enzyme is relatively stable, its activity is affected by numerous factors, including pressure. In this work, hydrostatic pressure dependence of the intrinsic tryptophan fluorescence in native and salted human butyrylcholinesterase was studied up to the maximum pressure at ambient temperature of about 1200MPa. A correlated large shift toward long wavelengths and broadening observed at pressures between 200 and 700MPa was interpreted as due to high pressure-induced denaturation of the protein, leading to an enhanced exposure of tryptophan residues into polar solvent environment. This transient process in native butyrylcholinesterase presumably involves conformational changes of the enzyme at both tertiary and secondary structure levels. Pressure-induced mixing of emitting local indole electronic transitions with quenching charge transfer states likely describes the accompanying fluorescence quenching that reveals different course from spectral changes. All the pressure-induced changes turned irreversible after passing a mid-point pressure of about 400+/-50MPa. Addition of either 0.1M ammonium sulphate (a kosmotropic salt) or 0.1M lithium thiocyanate (a chaotropic salt) to native enzyme similarly destabilized its structure.
ESTHER : Kangur_2019_Biochim.Biophys.Acta.Proteins.Proteom_1867_107
PubMedSearch : Kangur_2019_Biochim.Biophys.Acta.Proteins.Proteom_1867_107
PubMedID: 30414450

Title : A new sensitive spectrofluorimetric method for measurement of activity and kinetic study of cholinesterases - Mukhametgalieva_2019_Biochim.Biophys.Acta.Proteins.Proteom__140270
Author(s) : Mukhametgalieva AR , Zueva IV , Aglyamova AR , Lushchekina SV , Masson P
Ref : Biochimica & Biophysica Acta Proteins Proteom , :140270 , 2019
Abstract : A new spectrofluorimetric method more sensitive than the Ellman method was developed for determination of both acetylcholinesterase and butyrylcholinesterase activity and for kinetic analysis of these enzymes and their mutants. Two selected mutants of human butyrylcholinesterase (E197Q and E197G) were included in this work. As for the Ellman's method, substrates are thiocholine esters, but the chromogenic reagent, DTNB (dithio-bisnitro benzoic acid) is replaced by a fluorogenic probe, "Calbiochem Probe IV", (3-(7-Hydroxy-2-oxo-2H-chromen-3-ylcarbamoyl)acrylic acid methylester). Compared to the classical Ellman's method, the sensitivity of this new spectrofluorimetric assay is 2 orders of magnitude higher. The method allows measurement of activity in media containing <10(-11)M of cholinesterase active sites at low substrate concentrations, either under first order conditions, [S]<
ESTHER : Mukhametgalieva_2019_Biochim.Biophys.Acta.Proteins.Proteom__140270
PubMedSearch : Mukhametgalieva_2019_Biochim.Biophys.Acta.Proteins.Proteom__140270
PubMedID: 31518689

Title : 6-Methyluracil derivatives as peripheral site ligand-hydroxamic acid conjugates: Reactivation for paraoxon-inhibited acetylcholinesterase - Semenov_2019_Eur.J.Med.Chem_185_111787
Author(s) : Semenov VE , Zueva IV , Lushchekina SV , Lenina OA , Gubaidullina LM , Saifina LF , Shulaeva MM , Kayumova RM , Saifina AF , Gubaidullin AT , Kondrashova SA , Latypov SK , Masson P , Petrov KA
Ref : Eur Journal of Medicinal Chemistry , 185 :111787 , 2019
Abstract : New uncharged conjugates of 6-methyluracil derivatives with imidazole-2-aldoxime and 1,2,4-triazole-3-hydroxamic acid units were synthesized and studied as reactivators of organophosphate-inhibited cholinesterase. Using paraoxon (POX) as a model organophosphate, it was shown that 6-methyluracil derivatives linked with hydroxamic acid are able to reactivate POX-inhibited human acetylcholinesterase (AChE) in vitro. The reactivating efficacy of one compound (5b) is lower than that of pyridinium-2-aldoxime (2-PAM). Meanwhile, unlike 2-PAM, in vivo study showed that the lead compound 5b is able: (1) to reactivate POX-inhibited AChE in the brain; (2) to decrease death of neurons and, (3) to prevent memory impairment in rat model of POX-induced neurodegeneration.
ESTHER : Semenov_2019_Eur.J.Med.Chem_185_111787
PubMedSearch : Semenov_2019_Eur.J.Med.Chem_185_111787
PubMedID: 31675511

Title : New evidence for dual binding site inhibitors of acetylcholinesterase as improved drugs for treatment of Alzheimer's disease - Zueva_2019_Neuropharmacol_155_131
Author(s) : Zueva IV , Dias J , Lushchekina SV , Semenov VE , Mukhamedyarov MA , Pashirova TN , Babaev VM , Nachon F , Petrova N , Nurullin LF , Zakharova LY , Ilyin VI , Masson P , Petrov KA
Ref : Neuropharmacology , 155 :131 , 2019
Abstract : Profound synaptic dysfunction contributes to early loss of short-term memory in Alzheimer's disease. This study was set up to analyze possible neuroprotective effects of two dual binding site inhibitors of acetylcholinesterase (AChE), a new 6-methyluracil derivative, C-35, and the clinically used inhibitor donepezil. Crystal structure of the complex between human AChE and C-35 revealed tight contacts of ligand along the enzyme active site gorge. Molecular dynamics simulations indicated that the external flexible part of the ligand establishes multiple transient interactions with the enzyme peripheral anionic site. Thus, C-35 is a dual binding site inhibitor of AChE. In transgenic mice, expressing a chimeric mouse/human amyloid precursor protein and a human presenilin-1 mutant, C-35 (5mg/kg, i.p) and donepezil (0.75mg/kg, i.p) partially reversed synapse loss, decreased the number of amyloid plaques, and restored learning and memory. To separate temporal symptomatic therapeutic effects, associated with the increased lifetime of acetylcholine in the brain, from possible disease-modifying effect, an experimental protocol based on drug withdrawal from therapy was performed. When administration of C-35 and donepezil was terminated three weeks after the trial started, animals that were receiving C-35 showed a much better ability to learn than those who received vehicle or donepezil. Our results provide additional evidence that dual binding site inhibitors of AChE have Alzheimer's disease-modifying action.
ESTHER : Zueva_2019_Neuropharmacol_155_131
PubMedSearch : Zueva_2019_Neuropharmacol_155_131
PubMedID: 31132435
Gene_locus related to this paper: human-ACHE

Title : The four-helix bundle in cholinesterase dimers: Structural and energetic determinants of stability - Novichkova_2019_Chem.Biol.Interact_13ChEPon_309_108699
Author(s) : Novichkova DA , Lushchekina SV , Dym O , Masson P , Silman I , Sussman JL
Ref : Chemico-Biological Interactions , 309 :108699 , 2019
Abstract : The crystal structures of truncated forms of cholinesterases provide good models for assessing the role of non-covalent interactions in dimer assembly in the absence of cross-linking disulfide bonds. These structures identify the four-helix bundle that serves as the interface for formation of acetylcholinesterase and butyrylcholinesterase dimers. Here we performed a theoretical comparison of the structural and energetic factors governing dimerization. This included identification of inter-subunit and intra-subunit hydrogen bonds and hydrophobic interactions, evaluation of solvent-accessible surfaces, and estimation of electrostatic contributions to dimerization. To reveal the contribution to dimerization of individual amino acids within the contact area, free energy perturbation alanine screening was performed. Markov state modelling shows that the loop between the alpha13 and alpha14 helices in BChE is unstable, and occupies 4 macro-states. The order of magnitude of mean first passage times between these macrostates is ~10(-8)s. Replica exchange molecular dynamics umbrella sampling calculations revealed that the free energy of human BChE dimerization is -15.5kcal/mol, while that for human AChE is -26.4kcal/mol. Thus, the C-terminally truncated human butyrylcholinesterase dimer is substantially less stable than that of human acetylcholinesterase. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:1.
ESTHER : Novichkova_2019_Chem.Biol.Interact_13ChEPon_309_108699
PubMedSearch : Novichkova_2019_Chem.Biol.Interact_13ChEPon_309_108699
PubMedID: 31202688

Title : Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad - Grigorenko_2019_Chem.Biol.Interact_13ChEPon_306_138
Author(s) : Grigorenko BL , Novichkova DA , Lushchekina SV , Zueva IV , Schopfer LM , Nemukhin AV , Varfolomeev SD , Lockridge O , Masson P
Ref : Chemico-Biological Interactions , 306 :138 , 2019
Abstract : A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198.H438.E325) was replaced by S198.H438.N322E in silico. Molecular dynamics for 1.5 mus and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198.H438.N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.
ESTHER : Grigorenko_2019_Chem.Biol.Interact_13ChEPon_306_138
PubMedSearch : Grigorenko_2019_Chem.Biol.Interact_13ChEPon_306_138
PubMedID: 31009643

Title : Time-course of human cholinesterases-catalyzed competing substrate kinetics - Mukhametgalieva_2019_Chem.Biol.Interact_310_108702
Author(s) : Mukhametgalieva AR , Aglyamova AR , Lushchekina SV , Golicnik M , Masson P
Ref : Chemico-Biological Interactions , 310 :108702 , 2019
Abstract : Competing substrate kinetic analysis of human butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) from the time-course of enzyme-catalyzed substrate hydrolysis, using spectrophotometric assays is described. This study is based on the use of a chromogenic reporter "visible" substrate (substrate A), whose complete hydrolysis time course is retarded by a competing "invisible" substrate (substrate B). For BChE, four visible substrates were used, two thiocholine esters, benzoylthiocholine and butyrylthiocholine, and two aryl-acylamides, o-nitro trifluoro acetaminide and 3-(acetamido)-N,N,N-trimethylanilinium. Three different competing invisible substrates were used, phenyl acetate, acetylcholine and butyrylcholine. For AChE, two visible substrates were used, acetylthiocholine and 3-(acetamido)-N,N,N-trimethylanilinium. For AChE, acetylcholine was competing with visible substrates. The ratio (R) of bimolecular rate constants, kcat/Km, for all couples of substrates, invisible/visible (B/A) covered all possible limit situations, R << 1, R approximately 1 and R >> 1. The kinetic approach, based on the method developed by Golicnik and Masson allowed determination of binding and catalytic parameters of cholinesterases for both visible and invisible substrates. This analysis was applied to michaelian and non-michaelian catalytic behaviors (activation and inhibition by excess substrate). Reevaluation of catalytic parameters obtained for acetylcholine and butyrylcholine more than 50 years ago was made. The method is fast, reliable, and particularly suitable for poorly soluble substrates and for substrates B when no direct spectrophotometric assays exist. Moreover, replacing substrate B by a reversible inhibitor, mechanism of cholinesterase inhibition was possible to study. It is therefore, useful for screening libraries of new substrates and inhibitors, and/or screening of new cholinesterase mutants. This method can be applied to any other enzymes.
ESTHER : Mukhametgalieva_2019_Chem.Biol.Interact_310_108702
PubMedSearch : Mukhametgalieva_2019_Chem.Biol.Interact_310_108702
PubMedID: 31247192

Title : Water structure changes in oxime-mediated reactivation process of phosphorylated human acetylcholinesterase - Zueva_2018_Biosci.Rep_38_
Author(s) : Zueva IV , Lushchekina SV , Masson P
Ref : Bioscience Reports , 38 : , 2018
Abstract : The role of water in oxime-mediated reactivation of phosphylated cholinesterases (ChEs) has been asked with recurrence. To investigate oximate water structure changes in this reaction, reactivation of paraoxon-inhibited human acetylcholinesterase (AChE) was performed by the oxime asoxime (HI-6) at different pH in the presence and absence of lyotropic salts: a neutral salt (NaCl), a strong chaotropic salt (LiSCN) and strong kosmotropic salts (ammonium sulphate and phosphate HPO(4)(2-)). At the same time, molecular dynamic (MD) simulations of enzyme reactivation under the same conditions were performed over 100 ns. Reactivation kinetics showed that the low concentration of chaotropic salt up to 75 mM increased the percentage of reactivation of diethylphosphorylated AChE whereas kosmotropic salts lead only to a small decrease in reactivation. This indicates that water-breaker salt induces destructuration of water molecules that are electrostricted around oximate ions. Desolvation of oximate favors nucleophilic attack on the phosphorus atom. Effects observed at high salt concentrations (>100 mM) result either from salting-out of the enzyme by kosmotropic salts (phosphate and ammonium sulphate) or denaturing action of chaotropic LiSCN. MDs simulations of diethylphosphorylated hAChE complex with HI-6 over 100 ns were performed in the presence of 100 mM (NH(4))(2)SO(4) and 50 mM LiSCN. In the presence of LiSCN, it was found that protein and water have a higher mobility, i.e. water is less organized, compared with the ammonium sulphate system. LiSCN favors protein solvation (hydrophobic hydration) and breakage of elelectrostricted water molecules around of oximate ion. As a result, more free water molecules participated to reaction steps accompanying oxime-mediated dephosphorylation.
ESTHER : Zueva_2018_Biosci.Rep_38_
PubMedSearch : Zueva_2018_Biosci.Rep_38_
PubMedID: 29773682

Title : Combination delivery of two oxime-loaded lipid nanoparticles: Time-dependent additive action for prolonged rat brain protection - Pashirova_2018_J.Control.Release_290_102
Author(s) : Pashirova TN , Braiki A , Zueva IV , Petrov KA , Babaev VM , Burilova EA , Samarkina DA , Rizvanov IK , Souto EB , Jean L , Renard PY , Masson P , Zakharova LY , Sinyashin OG
Ref : J Control Release , 290 :102 , 2018
Abstract : A novel approach for brain protection against poisoning by organophosphorus agents is developed based on the combination treatment of dual delivery of two oximes. Pralidoxime chloride (2-PAM) and a novel reactivator, 6-(5-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)pentyl)-3-hydroxy picolinaldehyde oxime (3-HPA), have been loaded in solid-lipid nanoparticles (SLNs) to offer distinct release profile and systemic half-life for both oximes. To increase the therapeutic time window of both oximes, SLNs with two different compartments were designed to load each respective drug. Oxime-loaded SLNs of hydrodynamic diameter between 100 and 160nm and negative zeta potential (-30 to -25mV) were stable for a period of 10months at 4 degrees C. SLNs displayed longer circulation time in the bloodstream compared to free 3-HPA and free 2-PAM. Oxime-loaded SLNs were suitable for intravenous (iv) administration. Paraoxon-poisoned rats (0.8xLD50) were treated with 3-HPA-loaded SLNs and 2-PAM+3-HPA-loaded SLNs at the dose of 3-HPA and 2-PAM of 5mg/kg. Brain AChE reactivation up to 30% was slowly achieved in 5h after administration of 3-HPA-SLNs. For combination therapy with two oximes, a time-dependent additivity and increased reactivation up to 35% were observed.
ESTHER : Pashirova_2018_J.Control.Release_290_102
PubMedSearch : Pashirova_2018_J.Control.Release_290_102
PubMedID: 30308259

Title : Catalytic bioscavengers against organophosphorus agents: mechanistic issues of self-reactivating cholinesterases - Lushchekina_2018_Toxicology_409_91
Author(s) : Lushchekina S , Masson P
Ref : Toxicology , 409 :91 , 2018
Abstract : Catalytic bioscavengers are the second-generation bioscavengers. These biopharmaceuticals are intended to degrade toxic organophosphorus agents on the skin for decontamination or in the bloodstream for pre-treatment and post-exposure treatment of organophosphate poisoning. Because catalytic degradation has to be fast, their catalytic efficiency has to be as high as possible (kcat/Km>10(6) M(-1) min(-1)). Certain evolved mammalian paraoxonases and bacterial phosphotriesterases already fulfill this requirement. To be of interest, the catalytic activity of certain enzymes has to be increased by several orders of magnitude. This can be reached by computer-redesign or directed evolution existing enzymes, and alternatively, combinational strategies. The present paper focuses on the better understanding of catalytic mechanisms of cholinesterase inhibition, aging and reactivation and how this knowledge serves the rational design of novel catalytic bioscavengers based on cholinesterase structure.
ESTHER : Lushchekina_2018_Toxicology_409_91
PubMedSearch : Lushchekina_2018_Toxicology_409_91
PubMedID: 30056174

Title : C-547, a 6-methyluracil derivative with long-lasting binding and rebinding on acetylcholinesterase: Pharmacokinetic and pharmacodynamic studies - Petrov_2018_Neuropharmacol_131_304
Author(s) : Petrov KA , Zueva IV , Kovyazina IV , Sedov I , Lushchekina SV , Kharlamova AD , Lenina O , Koshkin S , Shtyrlin Y , Nikolsky EE , Masson P
Ref : Neuropharmacology , 131 :304 , 2018
Abstract : C-547, a potent slow-binding inhibitor of acetylcholinesterase (AChE) was intravenously administered to rat (0.05mg/kg). Pharmacokinetic profiles were determined in blood and different organs: extensor digitorum longus muscle, heart, liver, lungs and kidneys as a function of time. Pharmacokinetics (PK) was studied using non-compartmental and compartmental analyses. A 3-compartment model describes PK in blood. Most of injected C-547 binds to albumin in the bloodstream. The steady-state volume of distribution (3800ml/kg) is 15 times larger than the distribution volume, indicating a good tissue distribution. C-547 is slowly eliminated (kel=0.17 h(-1); T1/2=4h) from the bloodstream. Effect of C-547 on animal model of myasthenia gravis persists for more than 72h, even though the drug is not analytically detectable in the blood. A PK/PD model was built to account for such a pharmacodynamical (PD) effect. Long-lasting effect results from micro-PD mechanisms: the slow-binding nature of inhibition, high affinity for AChE and long residence time on target at neuromuscular junction (NMJ). In addition, NMJ spatial constraints i.e. high concentration of AChE in a small volume, and slow diffusion rate of free C-547 out of NMJ, make possible effective rebinding of ligand. Thus, compared to other cholinesterase inhibitors used for palliative treatment of myasthenia gravis, C-547 is the most selective drug, displays a slow pharmacokinetics, and has the longest duration of action. This makes C-547 a promising drug leader for treatment of myasthenia gravis, and a template for development of other drugs against neurological diseases and for neuroprotection.
ESTHER : Petrov_2018_Neuropharmacol_131_304
PubMedSearch : Petrov_2018_Neuropharmacol_131_304
PubMedID: 29277489

Title : Purification of recombinant human butyrylcholinesterase on Hupresin(R) - Lockridge_2018_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1102-1103_109
Author(s) : Lockridge O , David E , Schopfer LM , Masson P , Brazzolotto X , Nachon F
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 1102-1103 :109 , 2018
Abstract : Affinity chromatography on procainamide-Sepharose has been an important step in the purification of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) since its introduction in 1978. The procainamide affinity gel has limitations. In the present report a new affinity gel called Hupresin(R) was evaluated for its ability to purify truncated, recombinant human butyrylcholinesterase (rHuBChE) expressed in a stably transfected Chinese Hamster Ovary cell line. We present a detailed example of the purification of rHuBChE secreted into 3940mL of serum-free culture medium. The starting material contained 13,163units of BChE activity (20.9mg). rHuBChE was purified to homogeneity in a single step by passage over 82mL of Hupresin(R) eluted with 0.1M tetramethylammonium bromide in 20mM TrisCl pH7.5. The fraction with the highest specific activity of 630units/mg contained 11mg of BChE. Hupresin(R) is superior to procainamide-Sepharose for purification of BChE, but is not suitable for purifying native AChE because Hupresin(R) binds AChE so tightly that AChE is not released with buffers, but is desorbed with denaturing solvents such as 50% acetonitrile or 1% trifluoroacetic acid. Procainamide-Sepharose will continue to be useful for purification of AChE.
ESTHER : Lockridge_2018_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1102-1103_109
PubMedSearch : Lockridge_2018_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1102-1103_109
PubMedID: 30384187

Title : 3D structure of the natural tetrameric form of human butyrylcholinesterase as revealed by cryoEM, SAXS and MD - Boyko_2018_Biochimie_156_196
Author(s) : Boyko KM , Baymukhametov TN , Chesnokov YM , Hons M , Lushchekina SV , Konarev PV , Lipkin AV , Vasiliev AL , Masson P , Popov VO , Kovalchuk MV
Ref : Biochimie , 156 :196 , 2018
Abstract : Human plasma butyrylcholinesterase (BChE) is an endogenous bioscavenger that hydrolyzes numerous medicamentous and poisonous esters and scavenges potent organophosphorus nerve agents. BChE is thus a marker for the diagnosis of OP poisoning. It is also considered a therapeutic target against Alzheimer's disease. Although the X-ray structure of a partially deglycosylated monomer of human BChE was solved 15 years ago, all attempts to determine the 3D structure of the natural full-length glycosylated tetrameric human BChE have been unsuccessful so far. Here, a combination of three complementary structural methods-single-particle cryo-electron microscopy, molecular dynamics and small-angle X-ray scattering-were implemented to elucidate the overall structural and spatial organization of the natural tetrameric human plasma BChE. A 7.6A cryoEM map clearly shows the major features of the enzyme: a dimer of dimers with a nonplanar monomer arrangement, in which the interconnecting super helix complex PRAD-(WAT)4-peptide C-terminal tail is located in the center of the tetramer, nearly perpendicular to its plane, and is plunged deep between the four subunits. Molecular dynamics simulations allowed optimization of the geometry of the molecule and reconstruction of the structural features invisible in the cryoEM density, i.e., glycan chains and glycan interdimer contact areas, as well as intermonomer disulfide bridges at the C-terminal tail. Finally, SAXS data were used to confirm the consistency of the obtained model with the experimental data. The tetramer organization of BChE is unique in that the four subunits are joined at their C-termini through noncovalent contacts with a short polyproline-rich peptide. This tetramer structure could serve as a model for the design of highly stable glycosylated tetramers.
ESTHER : Boyko_2018_Biochimie_156_196
PubMedSearch : Boyko_2018_Biochimie_156_196
PubMedID: 30385318

Title : Mixed cationic liposomes for brain delivery of drugs by the intranasal route: The acetylcholinesterase reactivator 2-PAM as encapsulated drug model - Pashirova_2018_Colloids.Surf.B.Biointerfaces_171_358
Author(s) : Pashirova TN , Zueva IV , Petrov KA , Lukashenko SS , Nizameev IR , Kulik NV , Voloshina AD , Almasy L , Kadirov MK , Masson P , Souto EB , Zakharova LY , Sinyashin OG
Ref : Colloids Surf B Biointerfaces , 171 :358 , 2018
Abstract : New mixed cationic liposomes based on L-alpha-phosphatidylcholine and dihexadecylmethylhydroxyethylammonium bromide (DHDHAB) were designed to overcome the BBB crossing by using the intranasal route. Synthesis and self-assembly of DHDHAB were performed. A low critical association concentration (0.01 mM), good solubilization properties toward hydrophobic dye Orange OT and antimicrobial activity against gram-positive bacteria Staphylococcus aureus (MIC=7.8 mug mL(-1)) and Bacillus cereus (MIC=7.8 mug mL(-1)), low hemolytic activities against human red blood cells (less than 10%) were achieved. Conditions for preparation of cationic vesicles and mixed liposomes with excellent colloidal stability at room temperature were determined. The intranasal administration of rhodamine B-loaded cationic liposomes was shown to increase bioavailability into the brain in comparison to the intravenous injection. The cholinesterase reactivator, 2-PAM, was used as model drug for the loading in cationic liposomes. 2-PAM-loaded cationic liposomes displayed high encapsulation efficiency ( approximately 90%) and hydrodynamic diameter close to 100 nm. Intranasally administered 2-PAM-loaded cationic liposomes were effective against paraoxon-induced acetylcholinesterase inhibition in the brain. 2-PAM-loaded liposomes reactivated 12 +/- 1% of brain acetylcholinesterase. This promising result opens the possibility to use marketed positively charged oximes in medical countermeasures against organophosphorus poisoning for reactivation of central acetylcholinesterase by implementing a non-invasive approach, via the "nose-brain" pathway.
ESTHER : Pashirova_2018_Colloids.Surf.B.Biointerfaces_171_358
PubMedSearch : Pashirova_2018_Colloids.Surf.B.Biointerfaces_171_358
PubMedID: 30059851

Title : Optimization of Cholinesterase-Based Catalytic Bioscavengers Against Organophosphorus Agents - Lushchekina_2018_Front.Pharmacol_9_211
Author(s) : Lushchekina SV , Schopfer LM , Grigorenko BL , Nemukhin AV , Varfolomeev SD , Lockridge O , Masson P
Ref : Front Pharmacol , 9 :211 , 2018
Abstract : Organophosphorus agents (OPs) are irreversible inhibitors of acetylcholinesterase (AChE). OP poisoning causes major cholinergic syndrome. Current medical counter-measures mitigate the acute effects but have limited action against OP-induced brain damage. Bioscavengers are appealing alternative therapeutic approach because they neutralize OPs in bloodstream before they reach physiological targets. First generation bioscavengers are stoichiometric bioscavengers. However, stoichiometric neutralization requires administration of huge doses of enzyme. Second generation bioscavengers are catalytic bioscavengers capable of detoxifying OPs with a turnover. High bimolecular rate constants (kcat/Km > 10(6) M(-1)min(-1)) are required, so that low enzyme doses can be administered. Cholinesterases (ChE) are attractive candidates because OPs are hemi-substrates. Moderate OP hydrolase (OPase) activity has been observed for certain natural ChEs and for G117H-based human BChE mutants made by site-directed mutagenesis. However, before mutated ChEs can become operational catalytic bioscavengers their dephosphylation rate constant must be increased by several orders of magnitude. New strategies for converting ChEs into fast OPase are based either on combinational approaches or on computer redesign of enzyme. The keystone for rational conversion of ChEs into OPases is to understand the reaction mechanisms with OPs. In the present work we propose that efficient OP hydrolysis can be achieved by re-designing the configuration of enzyme active center residues and by creating specific routes for attack of water molecules and proton transfer. Four directions for nucleophilic attack of water on phosphorus atom were defined. Changes must lead to a novel enzyme, wherein OP hydrolysis wins over competing aging reactions. Kinetic, crystallographic, and computational data have been accumulated that describe mechanisms of reactions involving ChEs. From these studies, it appears that introducing new groups that create a stable H-bonded network susceptible to activate and orient water molecule, stabilize transition states (TS), and intermediates may determine whether dephosphylation is favored over aging. Mutations on key residues (L286, F329, F398) were considered. QM/MM calculations suggest that mutation L286H combined to other mutations favors water attack from apical position. However, the aging reaction is competing. Axial direction of water attack is not favorable to aging. QM/MM calculation shows that F329H+F398H-based multiple mutants display favorable energy barrier for fast reactivation without aging.
ESTHER : Lushchekina_2018_Front.Pharmacol_9_211
PubMedSearch : Lushchekina_2018_Front.Pharmacol_9_211
PubMedID: 29593539

Title : Autoregulation of Acetylcholine Release and Micro-Pharmacodynamic Mechanisms at Neuromuscular Junction: Selective Acetylcholinesterase Inhibitors for Therapy of Myasthenic Syndromes - Petrov_2018_Front.Pharmacol_9_766
Author(s) : Petrov KA , Nikolsky EE , Masson P
Ref : Front Pharmacol , 9 :766 , 2018
Abstract : Neuromuscular junctions (NMJs) are directly involved into such indispensable to life processes as respiration and locomotion. However, motor nerve forms only one synaptic contact at each muscle fiber. This unique configuration requires specific properties and constrains to be effective. The very high density of acetylcholine receptors (AChRs) of muscle type in synaptic cleft and an excess of acetylcholine (ACh) released under physiological conditions make this synapse extremely reliable. Nevertheless, under pathological conditions such as myasthenia gravis and congenital myasthenic syndromes, the safety factor can be markedly reduced. Drugs used for short-term symptomatic therapy of these pathological states, cause partial inhibition of cholinesterases (ChEs). These enzymes catalyze the hydrolysis of ACh, thus terminate its action on AChRs. Extension of the lifetime of ACh molecules compensates muscular AChRs abnormalities and, consequently, rescues muscle contractions. In this mini review, we will first outline the functional organization of the NMJ, and then, consider the concept of the safety factor and how it may be changed. This will be followed by a look at autoregulation of ACh release that influences the safety factor of NMJs. Finally, we will consider the morphological features of NMJs as a putative reserve to increase effectiveness of pathological muscle weakness therapy by ChEs inhibitors due to opportunity to use micro-pharmacodynamic mechanisms.
ESTHER : Petrov_2018_Front.Pharmacol_9_766
PubMedSearch : Petrov_2018_Front.Pharmacol_9_766
PubMedID: 30050445

Title : Nanoparticle-Delivered 2-PAM for Rat Brain Protection against Paraoxon Central Toxicity - Pashirova_2017_ACS.Appl.Mater.Interfaces_9_16922
Author(s) : Pashirova TN , Zueva IV , Petrov KA , Babaev VM , Lukashenko SS , Rizvanov IK , Souto EB , Nikolsky EE , Zakharova LY , Masson P , Sinyashin OG
Ref : ACS Appl Mater Interfaces , 9 :16922 , 2017
Abstract : Solid lipid nanoparticles (SLNs) are among the most promising nanocarriers to target the blood-brain barrier (BBB) for drug delivery to the central nervous system (CNS). Encapsulation of the acetylcholinesterase reactivator, pralidoxime chloride (2-PAM), in SLNs appears to be a suitable strategy for protection against poisoning by organophosphorus agents (OPs) and postexposure treatment. 2-PAM-loaded SLNs were developed for brain targeting and delivery via intravenous (iv) administration. 2-PAM-SLNs displayed a high 2-PAM encapsulation efficiency ( approximately 90%) and loading capacity (maximum 30.8 +/- 1%). Drug-loaded particles had a mean hydrodynamic diameter close to 100 nm and high negative zeta potential (-54 to -15 mV). These properties contribute to improve long-term stability of 2-PAM-SLNs when stored both at room temperature (22 degrees C) and at 4 degrees C, as well as to longer circulation time in the bloodstream compared to free 2-PAM. Paraoxon-poisoned rats (2 x LD50) were treated with 2-PAM-loaded SLNs at a dose of 2-PAM of 5 mg/kg. 2-PAM-SLNs reactivated 15% of brain AChE activity. Our results confirm the potential use of SLNs loaded with positively charged oximes as a medical countermeasure both for protection against OPs poisoning and for postexposure treatment.
ESTHER : Pashirova_2017_ACS.Appl.Mater.Interfaces_9_16922
PubMedSearch : Pashirova_2017_ACS.Appl.Mater.Interfaces_9_16922
PubMedID: 28504886

Title : Cholinesterase reactivators and bioscavengers for pre- and post-exposure treatments of organophosphorus poisoning - Masson_2017_J.Neurochem_142 Suppl 2_26
Author(s) : Masson P , Nachon F
Ref : Journal of Neurochemistry , 142 Suppl 2 :26 , 2017
Abstract : Organophosphorus agents (OPs) irreversibly inhibit acetylcholinesterase (AChE) causing a major cholinergic syndrome. The medical counter-measures of OP poisoning have not evolved for the last 30 years with carbamates for pretreatment, pyridinium oximes-based AChE reactivators, antimuscarinic drugs and neuroprotective benzodiazepines for post-exposure treatment. These drugs ensure protection of peripheral nervous system and mitigate acute effects of OP lethal doses. However, they have significant limitations. Pyridostigmine and oximes do not protect/reactivate central AChE. Oximes poorly reactivate AChE inhibited by phosphoramidates. In addition, current neuroprotectants do not protect the central nervous system shortly after the onset of seizures when brain damage becomes irreversible. New therapeutic approaches for pre- and post-exposure treatments involve detoxification of OP molecules before they reach their molecular targets by administrating catalytic bioscavengers, among them phosphotriesterases are the most promising. Novel generation of broad spectrum reactivators are designed for crossing the blood-brain barrier and reactivate central AChE. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.
ESTHER : Masson_2017_J.Neurochem_142 Suppl 2_26
PubMedSearch : Masson_2017_J.Neurochem_142 Suppl 2_26
PubMedID: 28542985

Title : The C5 Variant of the Butyrylcholinesterase Tetramer Includes a Noncovalently Bound 60 kDa Lamellipodin Fragment - Schopfer_2017_Molecules_22_
Author(s) : Schopfer LM , Delacour H , Masson P , Leroy J , Krejci E , Lockridge O
Ref : Molecules , 22 : , 2017
Abstract : Humans with the C5 genetic variant of butyrylcholinesterase (BChE) have 30-200% higher plasma BChE activity, low body weight, and shorter duration of action of the muscle relaxant succinylcholine. The C5 variant has an extra, slow-moving band of BChE activity on native polyacrylamide gel electrophoresis. This band is about 60 kDa larger than wild-type BChE. Umbilical cord BChE in 100% of newborn babies has a C5-like band. Our goal was to identify the unknown, 60 kDa protein in C5. Both wild-type and C5 BChE are under the genetic control of two independent loci, the BCHE gene on Chr 3q26.1 and the RAPH1 (lamellipodin) gene on Chr 2q33. Wild-type BChE tetramers are assembled around a 3 kDa polyproline peptide from lamellipodin. Western blot of boiled C5 and cord BChE showed a positive response with an antibody to the C-terminus of lamellipodin. The C-terminal exon of lamellipodin is about 60 kDa including an N-terminal polyproline. We propose that the unknown protein in C5 and cord BChE is encoded by the last exon of the RAPH1 gene. In 90% of the population, the 60 kDa fragment is shortened to 3 kDa during maturation to adulthood, leaving only 10% of adults with C5 BChE.
ESTHER : Schopfer_2017_Molecules_22_
PubMedSearch : Schopfer_2017_Molecules_22_
PubMedID: 28661448
Gene_locus related to this paper: human-BCHE

Title : Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity - Terekhov_2017_Proc.Natl.Acad.Sci.U.S.A_114_2550
Author(s) : Terekhov SS , Smirnov IV , Stepanova AV , Bobik TV , Mokrushina YA , Ponomarenko NA , Belogurov AA, Jr. , Rubtsova MP , Kartseva OV , Gomzikova MO , Moskovtsev AA , Bukatin AS , Dubina MV , Kostryukova ES , Babenko VV , Vakhitova MT , Manolov AI , Malakhova MV , Kornienko MA , Tyakht AV , Vanyushkina AA , Ilina EN , Masson P , Gabibov AG , Altman S
Ref : Proc Natl Acad Sci U S A , 114 :2550 , 2017
Abstract : Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.
ESTHER : Terekhov_2017_Proc.Natl.Acad.Sci.U.S.A_114_2550
PubMedSearch : Terekhov_2017_Proc.Natl.Acad.Sci.U.S.A_114_2550
PubMedID: 28202731

Title : Application of Tetrameric Recombinant Human Butyrylcholinesterase as a Biopharmaceutical for Amelioration of Symptoms of Acute Organophosphate Poisoning - Terekhov_2017_Bull.Exp.Biol.Med_163_430
Author(s) : Terekhov SS , Palikov VA , Palikova YA , Dyachenko IA , Shamborant OG , Smirnov IV , Masson P , Gabibov AG
Ref : Bulletin of Experimental Biology & Medicine , 163 :430 , 2017
Abstract : We present a procedure for optimizing the expression of recombinant tetrameric butyrylcholinesterase that enables large-scale production with the yield >30 mg/liter (>90 mg/roller bottle). Intravenous injection of the preparation significantly increased survival and decreased the severity of symptoms of poisoning with paraoxon, an organophosphorus toxin.
ESTHER : Terekhov_2017_Bull.Exp.Biol.Med_163_430
PubMedSearch : Terekhov_2017_Bull.Exp.Biol.Med_163_430
PubMedID: 28853097

Title : Characterization of butyrylcholinesterase in bovine serum - Dafferner_2017_Chem.Biol.Interact_266_17
Author(s) : Dafferner AJ , Lushchekina SV , Masson P , Xiao G , Schopfer LM , Lockridge O
Ref : Chemico-Biological Interactions , 266 :17 , 2017
Abstract : Human butyrylcholinesterase (HuBChE) protects from nerve agent toxicity. Our goal was to determine whether bovine serum could be used as a source of BChE. Bovine BChE (BoBChE) was immunopurified from 100 mL fetal bovine serum (FBS) or 380 mL adult bovine serum by binding to immobilized monoclonal mAb2. Bound proteins were digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry. The results proved that FBS and adult bovine serum contain BoBChE. The concentration of BoBChE was estimated to be 0.04 mug/mL in FBS, and 0.03 mug/mL in adult bovine serum, values lower than the 4 mug/mL BChE in human serum. Nondenaturing gel electrophoresis showed that monoclonal mAb2 bound BoBChE but not bovine acetylcholinesterase (BoAChE) and confirmed that FBS contains BoBChE and BoAChE. Recombinant bovine BChE (rBoBChE) expressed in serum-free culture medium spontaneously reactivated from inhibition by chlorpyrifos oxon at a rate of 0.0023 min-1 (t1/2 = 301 min-1) and aged at a rate of 0.0138 min-1 (t1/2 = 50 min-1). Both BoBChE and HuBChE have 574 amino acids per subunit and 90% sequence identity. However, the apparent size of serum BoBChE and rBoBChE tetramers was much greater than the 340,000 Da of HuBChE tetramers. Whereas HuBChE tetramers include short polyproline rich peptides derived from lamellipodin, no polyproline peptides have been identified in BoBChE. We hypothesize that BoBChE tetramers use a large polyproline-rich protein to organize subunits into a tetramer and that the low concentration of BoBChE in serum is explained by limited quantities of an unidentified polyproline-rich protein.
ESTHER : Dafferner_2017_Chem.Biol.Interact_266_17
PubMedSearch : Dafferner_2017_Chem.Biol.Interact_266_17
PubMedID: 28189703
Gene_locus related to this paper: bovin-BCHE

Title : Dynamics of human acetylcholinesterase bound to non-covalent and covalent inhibitors shedding light on changes to the water network structure - Peters_2016_Phys.Chem.Chem.Phys_18_12992
Author(s) : Peters J , Martinez N , Trovaslet M , Scannapieco K , Koza MM , Masson P , Nachon F
Ref : Phys Chem Chem Phys , 18 :12992 , 2016
Abstract : We investigated the effects of non-covalent reversible and covalent irreversible inhibitors on human acetylcholinesterase and human butyrylcholinesterase. Remarkably a non-covalent inhibitor, Huperzine A, has almost no effect on the molecular dynamics of the protein, whereas the covalently binding nerve agent soman renders the molecular structure stiffer in its aged form. The modified movements were studied by incoherent neutron scattering on different time scales and they indicate a stabilization and stiffening of aged human acetylcholinesterase. It is not straightforward to understand the forces leading to this strong effect. In addition to the specific interactions of the adduct within the protein, some indications point towards an extensive water structure change for the aged conjugate as water Bragg peaks appeared at cryogenic temperature despite an identical initial hydration state for all samples. Such a change associated to an apparent increase in free water volume upon aging suggests higher ordering of the hydration shell that leads to the stiffening of protein. Thus, several additive contributions seem responsible for the improved flexibility or stiffening effect of the inhibitors rather than a single interaction.
ESTHER : Peters_2016_Phys.Chem.Chem.Phys_18_12992
PubMedSearch : Peters_2016_Phys.Chem.Chem.Phys_18_12992
PubMedID: 27109895

Title : Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma - Peng_2016_Chem.Biol.Interact_243_82
Author(s) : Peng H , Brimijoin S , Hrabovska A , Krejci E , Blake TA , Johnson RC , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 243 :82 , 2016
Abstract : Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals.
ESTHER : Peng_2016_Chem.Biol.Interact_243_82
PubMedSearch : Peng_2016_Chem.Biol.Interact_243_82
PubMedID: 26585590

Title : Sensing activity of cholinesterases through a luminescence response of the hexarhenium cluster complex [{Re6S8}(OH)6](4.) - Elistratova_2016_Analyst_141_4204
Author(s) : Elistratova JG , Mustafina AR , Brylev KA , Petrov KA , Shestopalov MA , Mironov YV , Babaev VM , Rizvanov IK , Masson P , Sinyashin OG
Ref : Analyst , 141 :4204 , 2016
Abstract : The present work describes a new method to sense cholinesterase-catalyzed hydrolysis of acetylcholine (ACh) through a luminescence response of the hexarhenium cluster complex [{Re6S8}(OH)6](4-). A proton released from acetylcholinesterase (AChE)- or butyrylcholinesterase (BuChE)-catalyzed hydrolysis of ACh results in time-resolved sensitization of cluster-centered luminescence. The sensitization results from protonation of apical hydroxo-groups of the cluster complex. The protonation is affected by a counter ion effect. Thus, optimal conditions for adequate sensing of acetic acid produced by ACh hydrolysis are highlighted. Time-resolved luminescence and pH measurements under conditions of AChE-catalyzed hydrolysis of ACh show a good correlation between the cluster-centered luminescence and pH-induced inhibition of AChE. The inhibition is not significant within the first two minutes of ACh hydrolysis. Thus, the luminescence response measured within two minutes is dependent on both substrate and enzyme concentrations, which fits with AChE and BuChE kinetics. The usability of cluster-centered luminescence for monitoring the concentration-dependent inhibition of AChE with irreversible inhibitors is demonstrated, using a carbamylating agent, pyridostigmine bromide, as a model.
ESTHER : Elistratova_2016_Analyst_141_4204
PubMedSearch : Elistratova_2016_Analyst_141_4204
PubMedID: 27169678

Title : Understanding the non-catalytic behavior of human butyrylcholinesterase silent variants: comparison of wild-type enzyme, catalytically active Ala328Cys mutant, and silent Ala328Asp variant - Lushchekina_2016_Chem.Biol.Interact_259_223
Author(s) : Lushchekina SV , Nemukhin AV , Varfolomeev SD , Masson P
Ref : Chemico-Biological Interactions , 259 :223 , 2016
Abstract : Conformational dynamics of wild-type human butyrylcholinesterase (BChE), two mutants of residue Ala328, the catalytically active Ala328Cys, and the catalytically inactive (silent) Ala328Asp, and their interactions with butyrylcholine were studied. The aim was to understand the molecular mechanisms by which point mutations may lead to silent BChE variant or alter catalytic activity. Importance of BChE natural variants is due to medical consequences, i.e. prolonged apnea, following administration of the myorelaxant esters, succinylcholine and mivacurium. Comparison of molecular dynamics (MD) simulations for the three model systems showed that: 1) the active mutant Ala328Cys mutant has some changes in configuration of catalytic residues, which do not prevent binding of butyrylcholine to the active site; 2) in the naturally-occurring silent variant Ala328Asp, the Asp328 carboxylate may either form a salt bridge with Lys339 or a H-bond with His438. In the first case, the Omega-loop swings off the gorge, disrupting the pi-cation binding site and the catalytic triad. In the second case, binding of cationic substrates in the catalytic center is also impaired. MD simulations carried out in 0.15 M NaCl, close to physiological ionic strength conditions, favored the second situation. It was seen that Asp328 forms a H-bond with the catalytic triad His438, which in turn disrupts the catalytic machinery. Therefore, we concluded that the Ala328Asp variant is not catalytically active because of that dramatic event. Computational results, consistent with in vitro biochemical data and clinical observations, validate our MD approach.
ESTHER : Lushchekina_2016_Chem.Biol.Interact_259_223
PubMedSearch : Lushchekina_2016_Chem.Biol.Interact_259_223
PubMedID: 27062896

Title : Slow-binding inhibition of acetylcholinesterase by an alkylammonium derivative of 6-methyluracil: mechanism and possible advantages for myasthenia gravis treatment - Kharlamova_2016_Biochem.J_473_1225
Author(s) : Kharlamova AD , Lushchekina SV , Petrov KA , Kots ED , Nachon F , Villard-Wandhammer M , Zueva IV , Krejci E , Reznik VS , Zobov VV , Nikolsky EE , Masson P
Ref : Biochemical Journal , 473 :1225 , 2016
Abstract : Inhibition of human AChE (acetylcholinesterase) and BChE (butyrylcholinesterase) by an alkylammonium derivative of 6-methyluracil, C-547, a potential drug for the treatment of MG (myasthenia gravis) was studied. Kinetic analysis of AChE inhibition showed that C-547 is a slow-binding inhibitor of type B, i.e. after formation of the initial enzyme.inhibitor complex (Ki=140 pM), an induced-fit step allows establishment of the final complex (Ki*=22 pM). The estimated koff is low, 0.05 min(-1) On the other hand, reversible inhibition of human BChE is a fast-binding process of mixed-type (Ki=1.77 muM; Ki'=3.17 muM). The crystal structure of mouse AChE complexed with C-547 was solved at 3.13 A resolution. The complex is stabilized by cation-pi, stacking and hydrogen-bonding interactions. Molecular dynamics simulations of the binding/dissociation processes of C-547 and C-35 (a non-charged analogue) to mouse and human AChEs were performed. Molecular modelling on mouse and human AChE showed that the slow step results from an enzyme conformational change that allows C-547 to cross the bottleneck in the active-site gorge, followed by formation of tight complex, as observed in the crystal structure. In contrast, the related non-charged compound C-35 is not a slow-binding inhibitor. It does not cross the bottleneck because it is not sensitive to the electrostatic driving force to reach the bottom of the gorge. Thus C-547 is one of the most potent and selective reversible inhibitors of AChE with a long residence time, tau=20 min, longer than for other reversible inhibitors used in the treatment of MG. This makes C-547 a promising drug for the treatment of this disease.
ESTHER : Kharlamova_2016_Biochem.J_473_1225
PubMedSearch : Kharlamova_2016_Biochem.J_473_1225
PubMedID: 26929400
Gene_locus related to this paper: mouse-ACHE

Title : Emergence of catalytic bioscavengers against organophosphorus agents - Masson_2016_Chem.Biol.Interact_259_319
Author(s) : Masson P , Lushchekina SV
Ref : Chemico-Biological Interactions , 259 :319 , 2016
Abstract : Bioscavengers are an effective alternative approach for pre- and post-exposure treatments of nerve agent (NA) poisoning. Bioscavengers are natural or recombinant enzymes, reactive proteins, and antibodies that neutralize NAs before they reach their physiological targets. They are administered by injection (protein or gene delivery vector) and react with NAs in the bloodstream. Other ways of delivery can be used: inhalation for pulmonary delivery, topical creams for skin protection, etc. Operational bioscavengers must be producible at low cost, not susceptible to induce immune response and adverse effects, and stable in the bloodstream, upon storage, and under field conditions. First generation bioscavengers, cholinesterases and carboxylesterases, are stoichiometric bioscavengers. However, stoichiometric neutralization of NAs needs administration of huge doses of costly biopharmaceuticals. Second generation bioscavengers are catalytic bioscavengers. These are capable of detoxifying organophosphates regeneratively. By virtue of high turnover, much lower doses are needed for rapid neutralization of toxicants. The most promising catalytic bioscavengers are evolved mutants of phosphotriesterases (bacterial enzymes, mammalian paraoxonases), displaying enantiomeric preference for toxic NA isomers. However, engineering of cholinesterases, carboxylesterases, prolidases and other enzymes, e.g. phosphotriesterases-lactonases from extremophiles is of interest. In particular, association of cholinesterase mutants (not susceptible to age after phosphylation) with fast-reactivating oximes leads to pseudocatalytic bioscavengers. Thus, catalytic and pseudocatalytic bioscavengers are an improvement of bioscavenger-based medical countermeasures in terms of efficacy and cost.
ESTHER : Masson_2016_Chem.Biol.Interact_259_319
PubMedSearch : Masson_2016_Chem.Biol.Interact_259_319
PubMedID: 26899146

Title : Current and emerging strategies for organophosphate decontamination: special focus on hyperstable enzymes - Jacquet_2016_Environ.Sci.Pollut.Res.Int_23_8200
Author(s) : Jacquet P , Daude D , Bzdrenga J , Masson P , Elias M , Chabriere E
Ref : Environ Sci Pollut Res Int , 23 :8200 , 2016
Abstract : Organophosphorus chemicals are highly toxic molecules mainly used as pesticides. Some of them are banned warfare nerve agents. These compounds are covalent inhibitors of acetylcholinesterase, a key enzyme in central and peripheral nervous systems. Numerous approaches, including chemical, physical, and biological decontamination, have been considered for developing decontamination methods against organophosphates (OPs). This work is an overview of both validated and emerging strategies for the protection against OP pollution with special attention to the use of decontaminating enzymes. Considerable efforts have been dedicated during the past decades to the development of efficient OP degrading biocatalysts. Among these, the promising biocatalyst SsoPox isolated from the archaeon Sulfolobus solfataricus is emphasized in the light of recently published results. This hyperthermostable enzyme appears to be particularly attractive for external decontamination purposes with regard to both its catalytic and stability properties.
ESTHER : Jacquet_2016_Environ.Sci.Pollut.Res.Int_23_8200
PubMedSearch : Jacquet_2016_Environ.Sci.Pollut.Res.Int_23_8200
PubMedID: 26832878

Title : Human butyrylcholinesterase polymorphism: Molecular modeling - Lushchekina_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S80
Author(s) : Lushchekina SV , Delacour H , Lockridge O , Masson P
Ref : Int J Risk Saf Med , 27 Suppl 1 :S80 , 2015
Abstract : BACKGROUND: Prolonged apnoea following injection of ester-containing myoralaxants was first described in 1953. Because a large part of administered succinylcholine is shortly hydrolyzed by plasma butyrylcholinesterase (BChE) under normal conditions, prolonged apnoea was attributed to deficiency in BChE. It was found that BChE deficiency was due to genetic variations. Human BChE gene shows a large polyallelism. About 75 natural mutations of the BCHE gene have been documented so far [1]. Most of them cause alteration in BChE activity through point mutation effect on catalytic activity. Frame shifts and stop codons may also affect expression, or cause truncations in the sequence. OBJECTIVE: Recently, two novel BChE "silent" variants, Val204Asp [2] and Ala34Val [3], causing prolonged neuromuscular block after administration of mivacurium, were discovered. Mutations were genetically and kinetically characterized. The aim of the current study was to understand how these mutations determine "silent" phenotype.
METHODS: Molecular dynamics studies were carried out with NAMD 2.9 software at the Lomonosov supercomputer. Charmm 36 force field was used, periodical boundary conditions, 1 atm pressure, 298 K. 100 ns molecular dynamics runs were performed for the wild-type BChE and its mutants Val204Asp and Ala34Val.
RESULTS: Unlike wild-type BChE, which retained its operative catalytic triad through the whole MD simulation, the catalytic triad of mutants was disrupted, making chemical step impossible. Val204Asp mutation leads to reorganization of hydrogen bonding network around the catalytic triad, which in turn increases the distance between catalytic residue main chains. Mutation Ala34Val, located on the protein surface, leads to increased fluctuations in the Omega-loop and subsequent disruption of the gorge structure, including disruption of the catalytic triad and formation of new hydrogen bonds involving catalytic center residues.
CONCLUSIONS: Comparative study of the "silent" Ala328Asp mutant and the catalytically active mutant Ala328Cys shows that MD approach can discriminate between the differential effects of point mutations at a same position.
ESTHER : Lushchekina_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S80
PubMedSearch : Lushchekina_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S80
PubMedID: 26639724

Title : Molecular modeling of mechanism of action of anti-myasthenia gravis slow-binding inhibitor of acetylcholinesterase - Lushchekina_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S74
Author(s) : Lushchekina SV , Kots ED , Kharlamova AD , Petrov KA , Masson P
Ref : Int J Risk Saf Med , 27 Suppl 1 :S74 , 2015
Abstract : BACKGROUND: Myasthenia gravis (MG) is a chronic autoimmune neuromuscular disorder characterized by fluctuating weakness of voluntary skeletal muscles. The cause of autoimmune response is unknown and only symptomatic therapies for MG are currently available. Pharmacological correction of synaptic failure underlying MG, involves partial inhibition acetyl- and butyrylcholinesterase. Effectiveness of cholinesterase inhibitors in the symptomatic treatment of MG is based on their ability to potentiate the effects of acetylcholine by decreasing the rate of its enzymatic hydrolysis at neuromuscular junctions. Several new inhibitors of AChE were tested in animal model of MG and may be considered as valuable candidates for the treatment of pathological muscle weakness syndromes. In this study, we have investigated mechanisms of ChE inhibition by one of the most active 6-methyluracil derivatives (C547), as well as the possible benefits of using this compound for MG treatment compared to traditionally used pyridostigmine bromide.It was experimentally shown that C547 is a <<pseudo-irreversible>> slow-binding inhibitor of human AChE. Human BChE is reversibly inhibited by C547 with an affinity about 4 orders of magnitude lower than that of human AChE. Slow-binding inhibition of AChE leads to a lasting (over 24 hours) effect on the symptoms of muscle weakness in animal model of MG after a single administration of C547. OBJECTIVE: The aim of the present molecular modeling study was to reveal mechanism of AChE inhibition by C547 and elucidate its apparent <<pseudo-irreversibility>>.
METHODS: Two principle methods used in the present study were molecular docking and molecular dynamics (MD). Molecular docking was performed with Autodock 4.2.6 software, Lamarckian Genetic Algorithm to obtain structure of protein inhibitor complexes and Local Search for MD snapshots to compare relative binding affinity. For MD simulations NAMD 2.10 software with Charrm 36 force field was used, for the ligand C547 Charmm General Force Field was used, and missing parameters were obtained with quantum mechanical calculations. Unconstrained MD, steered MD (SMD) and free energy calculations with adaptive biasing force were performed.
RESULTS: During unconstrained MD, C547 very rapidly binded to the peripheral anionic site (PAS) of AChE. To pass the bottleneck, application of the external force was required (SMD). Both SMD modelling and free energy calculation revealed that after crossing the AChE bottleneck, C547 falls into very favorable position. At the same time the rupture of interactions as well as overcoming the bottleneck gates in the course of pulling out procedure requires application of much higher force than during the pulling-in process. This difference between binding and dissociating processes explains apparent <<pseudo-irreversibility>> of the inhibitor.
CONCLUSIONS: These findings are in good agreement with kinetics study showing that C-547 is a slow-binding inhibitor of type B, i.e. after rapid initial binding of inhibitor, the enzyme-inhibitor complex undergoes an isomerization step. Position obtained by SMD is in good agreement with X-ray data obtained by F. Nachon, IBS, France.
ESTHER : Lushchekina_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S74
PubMedSearch : Lushchekina_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S74
PubMedID: 26639721

Title : 6-Methyluracil derivatives as acetylcholinesterase inhibitors for treatment of Alzheimer's disease - Zueva_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S69
Author(s) : Zueva IV , Semenov VE , Mukhamedyarov MA , Lushchekina SV , Kharlamova AD , Petukhova EO , Mikhailov AS , Podyachev SN , Saifina LF , Petrov KA , Minnekhanova OA , Zobov VV , Nikolsky EE , Masson P , Reznik VS
Ref : Int J Risk Saf Med , 27 Suppl 1 :S69 , 2015
Abstract : BACKGROUND: Alzheimer's disease (AD) is the major age-related progressive neurodegenerative disorder. The brain of AD patients suffers from loss of cholinergic neurons and decreased number of synapses [1]. AD is caused by an imbalance between Abeta production and clearance, resulting in increased amount of Abeta in various forms [2]. Reduction of Abeta production and increasing clearance of Abeta pathogenic forms are key targets in the development of potential therapeutic agents for AD treatment. Unfortunately, only nosotropic approaches for treatment of AD are currently effective in humans. These approaches mainly focus on the inhibition of brain acetyl-cholinesterase (AChE) to increase lifetime of cerebral acetylcholine [3]. It is important to emphasize that AChE itself promotes the formation of Abeta fibrils in vitro and Abeta plaques in the cerebral cortex of transgenic mouse models of AD [4]. This property of AChE results from interaction between Abeta and the peripheral anionic site of the enzyme (PAS) [5]. Dual binding site inhibitors of both catalytic active site (CAS) and PAS can simultaneously improve cognition and slow down the rate of Abeta-induced neural degeneration. Unfortunately, the assortment of AChE PAS ligands is still extremely limited. OBJECTIVE: To study putative advantages of AChE non-charged PAS inhibitors based on 6-methyluracil derivatives for the treatment of Alzheimer's disease.
METHODS: In vitro studies. Concentration of drug producing 50% of AChE/BuChE activity inhibition (IC50) was measured using the method of Ellman et al. [6]. Toxicological experiments were performed using IP injection of the different compounds in mice. LD50, dose (in mg/kg) causing lethal effects in 50% of animals was taken as a criterion of toxicity [7]. The ability of compound to block in vitro AChE-induced Abeta1-40 aggregation was studied using a thioflavin T (ThT) fluorescent probe [8].In vivo biological assays. For in vivo blood-brain barrier permeation assay brains were removed 30 min after IP injection of LD50 dose of tested compound injection. The inhibitory potency was measured using the method of Ellman.Scopolamine and transgenic models of AD were used to evaluate the influence of compound 35 on spatial memory performance.Water solution of scopolamine was injected to mice (ip) 20 minutes before starting memory test during 14 days [9]. Mice were assigned to 7 groups, including 4 groups receiving injection (ip) of compound in different dosages, donepezil-treated mice (donepezil is conventionally used to treat Alzheimer's disease), positive and negative control groups. Double transgenic (APP/PS1) mice expressing a chimeric mouse/human amyloid precursor protein and a mutant of human presenilin-1 [10] were assigned to 4 groups, including transgenic animals injected (ip) with compound 35 or donepezil solution, positive (transgenes injected with water) and negative (wild-type mice) controls.To evaluate spatial memory performance, mice were trained on a reward alternation task using a conventional T-maze [11]. The criterion for a mouse having learned the rewarded alternation task was 3 consecutive days of at least 5 correct responses out of the 6 free trials.For beta-amyloid peptide load was evaluated quantitatively as a number and summary area of Thioflavine S fluorescent spots in cerebral cortex and hippocampal images using Image J program. Statistical analyses were performed using the Mann-Whitney test.
RESULTS: We evaluated the acute toxicity of the most active compounds. The most potent AChE inhibitor compound 35 (IC50 (AChE) = 5 +/- 0.5 nM) exhibited the lowest LD50 values (51 mg/kg) and inhibited brain AChE by more than 71 +/- 1%. Compound 35 at 10 nM, exhibited a significant (35 +/- 9%) inhibitory activity toward human AChE-induced Abeta aggregation.Scopolamine injection induced significant decrease in correct choice percentage in T-maze, as well as decrease in percentage of mice reaching criterion for learning the task by day 14. This memory deficit was relieved to some extent either by compound 35 (5 mg/kg) or donepezil (reference compound) treatment (0.75 mg/kg). Interestingly, higher doses of compound 35 (10 and 15 mg/kg) produced less therapeutic effect on spatial memory deficit.Group of APP/PS1 mice showed 3 times lower percentage of reaching behavioral criterion and lower percentage of correct choice in T-maze alternation task comparing to WT mice, whereas compound 35 (5 mg/kg) or Donepezil treatment effectively improved these parameters in APP/PS1 mice.Compound 35 treatment (5 mg/kg) during 14 days significantly reduced percentage of summary area and number of beta-amyloid peptide (betaAP) deposits visualized in sections of cerebral cortex, dentate gyrus, and hippocampal CA3 area in APP/PS1 mice. The most prominent reduction of betaAP load by compound 35 treatment was found in CA3 area and cerebral cortex. Meanwhile, Donepezil treatment (1 mg/kg) during 14 days significantly reduced betaAP load in cerebral cortex but not in dentate gyrus and CA3 area.
CONCLUSIONS: Experiments showed that the most potent AChE inhibitor compound 35 (6-methyluracil derivative) permeated the blood-brain barrier, improved working memory in the APP/PS1 transgenic mice and significantly reduced the number and area of Abeta plaques in the brain. Thus, compound 35 is a promising candidate as a bi-functional inhibitor of AChE for treatment of AD.
ESTHER : Zueva_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S69
PubMedSearch : Zueva_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S69
PubMedID: 26639718

Title : Luminescent silica nanoparticles for sensing acetylcholinesterase-catalyzed hydrolysis of acetylcholine - Mukhametshina_2015_Biosens.Bioelectron_77_871
Author(s) : Mukhametshina AR , Fedorenko SV , Zueva IV , Petrov KA , Masson P , Nizameev IR , Mustafina AR , Sinyashin OG
Ref : Biosensors & Bioelectronics , 77 :871 , 2015
Abstract : This work highlights the H-function of Tb(III)-doped silica nanoparticles in aqueous solutions of acetic acid as a route to sense acetylcholinesterase-catalyzed hydrolysis of acetylcholine (ACh). The H-function results from H+-induced quenching of Tb(III)-centered luminescence due to protonation of Tb(III) complexes located close to silica/water interface. The H-function can be turned on/switched off by the concentration of complexes within core or nanoparticle shell zones, by the silica surface decoration and adsorption of both organic and inorganic cations on silica surface. Results indicate the optimal synthetic procedure for making nanoparticles capable of sensing acetic acid produced by enzymatic hydrolysis of acetylcholine. The H-function of nanoparticles was determined at various concentrations of ACh and AChE. The measurements show experimental conditions for fitting the H-function to Michaelis-Menten kinetics. Results confirm that reliable fluorescent monitoring AChE-catalyzed hydrolysis of ACh is possible through the H-function properties of Tb(III)-doped silica nanoparticles.
ESTHER : Mukhametshina_2015_Biosens.Bioelectron_77_871
PubMedSearch : Mukhametshina_2015_Biosens.Bioelectron_77_871
PubMedID: 26516688

Title : Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: K values, binding pairs, and amino acid sequences - Peng_2015_Chem.Biol.Interact_240_336
Author(s) : Peng H , Brimijoin S , Hrabovska A , Targosova K , Krejci E , Blake TA , Johnson RC , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 240 :336 , 2015
Abstract : Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 degrees C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10-9 M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.
ESTHER : Peng_2015_Chem.Biol.Interact_240_336
PubMedSearch : Peng_2015_Chem.Biol.Interact_240_336
PubMedID: 26343001

Title : Chemical Polysialylation and In Vivo Tetramerization Improve Pharmacokinetic Characteristics of Recombinant Human Butyrylcholinesterase-Based Bioscavengers - Terekhov_2015_Acta.Naturae_7_136
Author(s) : Terekhov SS , Smirnov IV , Shamborant OG , Bobik TV , Ilyushin DG , Murashev AN , Dyachenko IA , Palikov VA , Knorre VD , Belogurov AA , Ponomarenko NA , Kuzina ES , Genkin DD , Masson P , Gabibov AG
Ref : Acta Naturae , 7 :136 , 2015
Abstract : Organophosphate toxins (OPs) are the most toxic low-molecular compounds. The extremely potent toxicity of OPs is determined by their specificity toward the nerve system. Human butyrylcholinesterase (hBChE) is a natural bioscavenger against a broad spectrum of OPs, which makes it a promising candidate for the development of DNA-encoded bioscavengers. The high values of the protective index observed for recombinant hBChE (rhBChE) make it appropriate for therapy against OP poisoning, especially in the case of highly toxic warfare nerve agents. Nevertheless, large-scale application of biopharmaceuticals based on hBChE is restricted due to its high cost and extremely rapid elimination from the bloodstream. In the present study, we examine two approaches for long-acting rhBChE production: I) chemical polysialylation and II) in-vivo tetramerization. We demonstrate that both approaches significantly improve the pharmacokinetic characteristics of rhBChE (more than 5 and 10 times, respectively), which makes it possible to use rhBChE conjugated with polysialic acids (rhBChE-CAO) and tetrameric rhBChE (4rhBChE) in the treatment of OP poisonings.
ESTHER : Terekhov_2015_Acta.Naturae_7_136
PubMedSearch : Terekhov_2015_Acta.Naturae_7_136
PubMedID: 26798501

Title : 6-Methyluracil Derivatives as Bifunctional Acetylcholinesterase Inhibitors for the Treatment of Alzheimer's Disease - Semenov_2015_ChemMedChem_10_1863
Author(s) : Semenov VE , Zueva IV , Mukhamedyarov MA , Lushchekina SV , Kharlamova AD , Petukhova EO , Mikhailov AS , Podyachev SN , Saifina LF , Petrov KA , Minnekhanova OA , Zobov VV , Nikolsky EE , Masson P , Reznik VS
Ref : ChemMedChem , 10 :1863 , 2015
Abstract : Novel 6-methyluracil derivatives with omega-(substituted benzylethylamino)alkyl chains at the nitrogen atoms of the pyrimidine ring were designed and synthesized. The numbers of methylene groups in the alkyl chains were varied along with the electron-withdrawing substituents on the benzyl rings. The compounds are mixed-type reversible inhibitors of cholinesterases, and some of them show remarkable selectivity for human acetylcholinesterase (hAChE), with inhibitory potency in the nanomolar range, more than 10 000-fold higher than that for human butyrylcholinesterase (hBuChE). Molecular modeling studies indicate that these compounds are bifunctional AChE inhibitors, spanning the enzyme active site gorge and binding to its peripheral anionic site (PAS). In vivo experiments show that the 6-methyluracil derivatives are able to penetrate the blood-brain barrier (BBB), inhibiting brain-tissue AChE. The most potent AChE inhibitor, 3 d (1,3-bis[5-(o-nitrobenzylethylamino)pentyl]-6-methyluracil), was found to improve working memory in scopolamine and transgenic APP/PS1 murine models of Alzheimer's disease, and to significantly decrease the number and area of beta-amyloid peptide plaques in the brain.
ESTHER : Semenov_2015_ChemMedChem_10_1863
PubMedSearch : Semenov_2015_ChemMedChem_10_1863
PubMedID: 26412714

Title : Pressure-induced molten globule state of human acetylcholinesterase: structural and dynamical changes monitored by neutron scattering - Marion_2015_Phys.Chem.Chem.Phys_17_3157
Author(s) : Marion J , Trovaslet M , Martinez N , Masson P , Schweins R , Nachon F , Trapp M , Peters J
Ref : Phys Chem Chem Phys , 17 :3157 , 2015
Abstract : We used small-angle neutron scattering (SANS) to study the effects of high hydrostatic pressure on the structure of human acetylcholinesterase (hAChE). At atmospheric pressure, our SANS results obtained on D11 at ILL (Grenoble, France) give a radius of gyration close to that calculated for a mixture of monomers, dimers and tetramers of the enzyme, suggesting a good agreement between hAChE crystal structure and its conformation in solution. Applying high pressure to the sample we found a global compression of about 11% of the enzyme up to a pressure of 900 bar and then again an extension up to 2.1 kbar indicating unfolding of the tertiary structure due to a molten globule (MG) state. On the other hand, we studied the influence of pressure up to 6 kbar on the dynamics of this enzyme, on the backscattering spectrometer IN13 at ILL. For the first time, we used elastic incoherent neutron scattering (EINS) to probe the differences between hAChE in its folded state (N), its high-pressure induced MG state and its unfolded state (U). Especially around the MG state at 1750 bar we found a significant increase in the dynamics, indicating a partial unfolding. A four-step-model is suggested to describe the changes in the protein.
ESTHER : Marion_2015_Phys.Chem.Chem.Phys_17_3157
PubMedSearch : Marion_2015_Phys.Chem.Chem.Phys_17_3157
PubMedID: 25515378

Title : A novel expression cassette delivers efficient production of exclusively tetrameric human butyrylcholinesterase with improved pharmacokinetics for protection against organophosphate poisoning - Terekhov_2015_Biochimie_118_51
Author(s) : Terekhov SS , Smirnov IV , Bobik TV , Shamborant OG , Zenkova MA , Chernolovskaya EL , Gladkikh DV , Murashev AN , Dyachenko IA , Palikov VA , Palikova YA , Knorre VD , Belogurov AA, Jr. , Ponomarenko NA , Blackburn GM , Masson P , Gabibov AG
Ref : Biochimie , 118 :51 , 2015
Abstract : Butyrylcholinesterase is a stoichiometric bioscavenger against poisoning by organophosphorus pesticides and nerve agents. The low level of expression and extremely rapid clearance of monomeric recombinant human butyrylcholinesterase (rhBChE) from bloodstream (t(1/2) approximately 2 min) limits its pharmaceutical application. Recently (Ilyushin at al., PNAS, 2013) we described a long-acting polysialylated recombinant butyrylcholinesterase (rhBChE-CAO), stable in the bloodstream, that protects mice against 4.2 LD50 of VR. Here we report a set of modifications of the initial rhBChE expression vector to improve stability of the enzyme in the bloodstream and increase its production in CHO cells by introducing in the expression cassette: (i) the sequence of the natural human PRAD-peptide in frame with rhBChE gene via "self-processing" viral F2A peptide under control of an hEF/HTLV promoter, and (ii) previously predicted in silico MAR 1-68 and MAR X-29 sequences. This provides fully tetrameric rhBChE (4rhBChE) at 70 mg/l, that displays improved pharmacokinetics (t(1/2) = 32 +/- 1.2 h, MRT = 43 +/- 2 h). 3D Fluorescent visualization and distribution of (125)I-labeled enzyme reveals similar low level 4rhBChE and rhBChE-CAO accumulation in muscle, fat, and brain. Administered 4rhBChE was mainly catabolized in the liver and breakdown products were excreted in kidney. Injection of 1.2 LD50 and 1.1 LD50 of paraoxon to BALB/c and knockout BChE-/- mice pre-treated with 4rhBChE (50 mg/kg) resulted in 100% and 78% survival, respectively, without perturbation of long-term behavior. In contrast, 100% mortality of non-pre-treated mice was observed. The high expression level of 4rhBChE in CHO cells permits consideration of this new expression system for manufacturing BChE as a biopharmaceutical.
ESTHER : Terekhov_2015_Biochimie_118_51
PubMedSearch : Terekhov_2015_Biochimie_118_51
PubMedID: 26239905

Title : Detection of cresyl phosphate-modified butyrylcholinesterase in human plasma for chemical exposure associated with aerotoxic syndrome - Schopfer_2014_Anal.Biochem_461C_17
Author(s) : Schopfer LM , Masson P , Lamourette P , Simon S , Lockridge O
Ref : Analytical Biochemistry , 461C :17 , 2014
Abstract : Flight crews complain of illness following a fume event in aircraft. A chemical in jet engine oil, the neurotoxicant tri-o-cresyl phosphate, after metabolic activation to cresyl saligenin phosphate makes a covalent adduct on butyrylcholinesterase (BChE). We developed a mass spectrometry method for detection of the cresyl phosphate adduct on human BChE as an indicator of exposure. Monoclonal mAb2, whose amino acid sequence is provided, was crosslinked to cyanogen bromide-activated Sepharose 4B and used to immunopurify plasma BChE treated with cresyl saligenin phosphate. BChE was released with acetic acid, digested with pepsin, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MSMS) on the Triple TOF 5600 mass spectrometer. Peptide FGES198AGAAS with an added mass of 170Da from cresyl phosphate on serine 198 (Ser198) was detected as parent ion 966.4Da. When characteristic daughter ions were monitored in the MSMS spectrum, the limit of detection was 0.1% cresyl saligenin phosphate inhibited plasma BChE. This corresponds to 2x10-9g in 0.5ml or 23x10-15moles of inhibited BChE in 0.5ml of plasma. In conclusion, a sensitive assay for exposure to tri-o-cresyl phosphate was developed. Laboratories that plan to use this method are cautioned that a positive result gives no proof that tri-o-cresyl phosphate is toxic at low levels.
ESTHER : Schopfer_2014_Anal.Biochem_461C_17
PubMedSearch : Schopfer_2014_Anal.Biochem_461C_17
PubMedID: 24892986

Title : Correlation of the dynamics of native human acetylcholinesterase and its inhibited huperzine A counterpart from sub-picoseconds to nanoseconds - Trapp_2014_J.R.Soc.Interface_11_
Author(s) : Trapp M , Tehei M , Trovaslet M , Nachon F , Martinez N , Koza MM , Weik M , Masson P , Peters J
Ref : J R Soc Interface , 11 : , 2014
Abstract : It is a long debated question whether catalytic activities of enzymes, which lie on the millisecond timescale, are possibly already reflected in variations in atomic thermal fluctuations on the pico- to nanosecond timescale. To shed light on this puzzle, the enzyme human acetylcholinesterase in its wild-type form and complexed with the inhibitor huperzine A were investigated by various neutron scattering techniques and molecular dynamics simulations. Previous results on elastic neutron scattering at various timescales and simulations suggest that dynamical processes are not affected on average by the presence of the ligand within the considered time ranges between 10 ps and 1 ns. In the work presented here, the focus was laid on quasi-elastic (QENS) and inelastic neutron scattering (INS). These techniques give access to different kinds of individual diffusive motions and to the density of states of collective motions at the sub-picoseconds timescale. Hence, they permit going beyond the first approach of looking at mean square displacements. For both samples, the autocorrelation function was well described by a stretched-exponential function indicating a linkage between the timescales of fast and slow functional relaxation dynamics. The findings of the QENS and INS investigation are discussed in relation to the results of our earlier elastic incoherent neutron scattering and molecular dynamics simulations.
ESTHER : Trapp_2014_J.R.Soc.Interface_11_
PubMedSearch : Trapp_2014_J.R.Soc.Interface_11_
PubMedID: 24872501

Title : Molecular Modeling Evidence for His438 Flip in the Mechanism of Butyrylcholinesterase Hysteretic Behavior - Lushchekina_2014_J.Mol.Neurosci_52_44
Author(s) : Lushchekina SV , Nemukhin AV , Varfolomeev SD , Masson P
Ref : Journal of Molecular Neuroscience , 52 :434 , 2014
Abstract : Cholinesterases display a hysteretic behavior with certain substrates and irreversible inhibitors. For years, this behavior has remained puzzling. However, several lines of evidence indicated that it is caused by perturbation of the catalytic triad and its water environment. In the present study, using molecular dynamics simulations of Ala328Cys BCHE mutant and wild-type BCHE in the absence and presence of a co-solvent (sucrose, glycerol), we provide evidence that hysteresis originates in a flip of the catalytic triad histidine (His438). This event is controlled by water molecules that interact with active site residues. The physiological significance of this phenomenon is still an issue.
ESTHER : Lushchekina_2014_J.Mol.Neurosci_52_44
PubMedSearch : Lushchekina_2014_J.Mol.Neurosci_52_44
PubMedID: 24310732

Title : Characterization of a Novel BCHE Silent Allele: Point Mutation (p.Val204Asp) Causes Loss of Activity and Prolonged Apnea with Suxamethonium - Delacour_2014_PLoS.One_9_e101552
Author(s) : Delacour H , Lushchekina SV , Mabboux I , Bousquet A , Ceppa F , Schopfer LM , Lockridge O , Masson P
Ref : PLoS ONE , 9 :e101552 , 2014
Abstract : Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of suxamethonium leading to the discovery of a novel BCHE variant (c.695T>A, p.Val204Asp). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation disrupts the catalytic triad and determines a "silent" phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with heterozygous atypical silent genotype. Electrophoretic analysis of plasma BChE of the proband and his mother showed that patient has a reduced amount of tetrameric enzyme in plasma and that minor fast-moving BChE components: monomer, dimer, and monomer-albumin conjugate are missing. Kinetic analysis showed that the p.Val204Asp/p.Asp70Gly-p.Ala539Thr BChE displays a pure Michaelian behavior with BTC as the substrate. Both catalytic parameters Km = 265 microM for BTC, two times higher than that of the atypical enzyme, and a low Vmax are consistent with the absence of activity against suxamethonium. Molecular dynamic (MD) simulations showed that the overall effect of the mutation p.Val204Asp is disruption of hydrogen bonding between Gln223 and Glu441, leading Ser198 and His438 to move away from each other with subsequent disruption of the catalytic triad functionality regardless of the type of substrate. MD also showed that the enzyme volume is increased, suggesting a pre-denaturation state. This fits with the reduced concentration of p.Ala204Asp/p.Asp70Gly-p.Ala539Thr tetrameric enzyme in the plasma and non-detectable fast moving-bands on electrophoresis gels.
ESTHER : Delacour_2014_PLoS.One_9_e101552
PubMedSearch : Delacour_2014_PLoS.One_9_e101552
PubMedID: 25054547

Title : Characterization of a novel butyrylcholinesterase point mutation (p.Ala34Val), silent with mivacurium - Delacour_2014_Biochem.Pharmacol_92_476
Author(s) : Delacour H , Lushchekina SV , Mabboux I , Ceppa F , Masson P , Schopfer LM , Lockridge O
Ref : Biochemical Pharmacology , 92 :476 , 2014
Abstract : Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivarcurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of mivacurium leading to the discovery of a novel BCHE variant (c.185C>T, p.Ala34Val). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation remote from the active center determines the "silent" phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with a heterozygous enzyme of type atypical/silent. Kinetic analysis with succinyldithiocholine (SCdTC) as the substrate showed that Ala34Val BChE was inactive against this substrate. However, with BTC, the mutant enzyme was active, displaying an unexpected activation by excess substrate. Competitive inhibition of BTC by mivacurium gave a Ki=1.35mM consistent with the lack of activity with the related substrate SCdTC, and with the clinical data. Molecular dynamic simulations revealed the mechanism by which mutation Ala34Val determines the silent phenotype: a chain of intramolecular events leads to disruption of the catalytic triad, so that His438 no longer interacts with Ser198, but instead forms hydrogen bonds either with residues Glu197 and Trp82, or peripheral site residue Tyr332. However, at high BTC concentration, initial binding of substrate to the peripheral site triggers restoration of a functional catalytic triad, and activity with BTC.
ESTHER : Delacour_2014_Biochem.Pharmacol_92_476
PubMedSearch : Delacour_2014_Biochem.Pharmacol_92_476
PubMedID: 25264279

Title : Effects of viscosity and osmotic stress on the reaction of human butyrylcholinesterase with cresyl saligenin phosphate, a toxicant related to aerotoxic syndrome: kinetic and molecular dynamics studies - Masson_2013_Biochem.J_454_387
Author(s) : Masson P , Lushchekina SV , Schopfer LM , Lockridge O
Ref : Biochemical Journal , 454 :387 , 2013
Abstract : CSP (cresyl saligenin phosphate) is an irreversible inhibitor of human BChE (butyrylcholinesterase) that has been involved in the aerotoxic syndrome. Inhibition under pseudo-first-order conditions is biphasic, reflecting a slow equilibrium between two enzyme states E and E'. The elementary constants for CSP inhibition of wild-type BChE and D70G mutant were determined by studying the dependence of inhibition kinetics on viscosity and osmotic pressure. Glycerol and sucrose were used as viscosogens. Phosphorylation by CSP is sensitive to viscosity and is thus strongly diffusion-controlled (kon approximately 108 M-1.min-1). Bimolecular rate constants (ki) are about equal to kon values, making CSP one of the fastest inhibitors of BChE. Sucrose caused osmotic stress because it is excluded from the active-site gorge. This depleted the active-site gorge of water. Osmotic activation volumes, determined from the dependence of ki on osmotic pressure, showed that water in the gorge of the D70G mutant is more easily depleted than that in wild-type BChE. This demonstrates the importance of the peripheral site residue Asp70 in controlling the active-site gorge hydration. MD simulations provided new evidence for differences in the motion of water within the gorge of wild-type and D70G enzymes. The effect of viscosogens/osmolytes provided information on the slow equilibrium Eright harpoon over left harpoonE', indicating that alteration in hydration of a key catalytic residue shifts the equilibrium towards E'. MD simulations showed that glycerol molecules that substitute for water molecules in the enzyme active-site gorge induce a conformational change in the catalytic triad residue His438, leading to the less reactive form E'.
ESTHER : Masson_2013_Biochem.J_454_387
PubMedSearch : Masson_2013_Biochem.J_454_387
PubMedID: 23782236

Title : PHOS-Select Iron Affinity Beads Enrich Peptides for the Detection of Organophosphorus Adducts on Albumin - Jiang_2013_Chem.Res.Toxicol_26_1917
Author(s) : Jiang W , Dubrovskii YA , Podolskaya EP , Murashko EA , Babakov VN , Nachon F , Masson P , Schopfer LM , Lockridge O
Ref : Chemical Research in Toxicology , 26 :1917 , 2013
Abstract : Albumin is covalently modified by organophosphorus toxicants (OP) on tyrosine 411, but less than 1% of albumin is modified in humans by lethal OP doses that inhibit 95% of plasma butyrylcholinesterase. A method that enriches OP-modified albumin peptides could aid analysis of low dose exposures. Soman or chlorpyrifos oxon treated human plasma was digested with pepsin. Albumin peptides were enriched by binding to Fe(3+) beads at pH 11 and eluted with pH 2.6 buffer. Similarly, mouse and guinea pig albumin modified by chlorpyrifos oxon were digested with pepsin and enriched by binding to Fe(3+) beads. Peptides were identified by MALDI-TOF/TOF mass spectrometry. PHOS-select iron affinity beads specifically enriched albumin peptides VRY411TKKVPQVST and LVRY411TKKVPQVST in a pepsin digest of human plasma. The unmodified as well as OP-modified peptides bound to the beads. The binding capacity of 500 muL of beads was the pepsin digest of 2.1 muL of human plasma. The limit of detection was 0.2% of OP-modified albumin peptide in 0.43 muL of plasma. Enrichment of OP-modified albumin peptides by binding to Fe(3+) beads is a method with potential application to diagnosis of OP pesticide and nerve agent exposure in humans, mice, and guinea pigs.
ESTHER : Jiang_2013_Chem.Res.Toxicol_26_1917
PubMedSearch : Jiang_2013_Chem.Res.Toxicol_26_1917
PubMedID: 24187955

Title : Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo - Ilyushin_2013_Proc.Natl.Acad.Sci.U.S.A_110_1243
Author(s) : Ilyushin DG , Smirnov IV , Belogurov AA, Jr. , Dyachenko IA , Zharmukhamedova T , Novozhilova TI , Bychikhin EA , Serebryakova MV , Kharybin ON , Murashev AN , Anikienko KA , Nikolaev EN , Ponomarenko NA , Genkin DD , Blackburn GM , Masson P , Gabibov AG
Ref : Proc Natl Acad Sci U S A , 110 :1243 , 2013
Abstract : The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD(50) of S-(2-(diethylamino)ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.
ESTHER : Ilyushin_2013_Proc.Natl.Acad.Sci.U.S.A_110_1243
PubMedSearch : Ilyushin_2013_Proc.Natl.Acad.Sci.U.S.A_110_1243
PubMedID: 23297221

Title : Mass spectrometry method to identify aging pathways of Sp- and Rp-tabun adducts on human butyrylcholinesterase based on the acid labile P-N bond - Jiang_2013_Toxicol.Sci_132_390
Author(s) : Jiang W , Cashman JR , Nachon F , Masson P , Schopfer LM , Lockridge O
Ref : Toxicol Sci , 132 :390 , 2013
Abstract : The phosphoramidate nerve agent tabun inhibits butyrylcholinesterase (BChE) and acetylcholinesterase by making a covalent bond on the active site serine. The adduct loses an alkyl group in a process called aging. The mechanism of aging of the tabun adduct is controversial. Some studies claim that aging proceeds through deamination, whereas crystal structure studies show aging by O-dealkylation. Our goal was to develop a method that clearly distinguishes between deamination and O-dealkylation. We began by studying the tetraisopropyl pyrophosphoramide adduct of BChE because this adduct has two P-N bonds. Mass spectra showed that the P-N bonds were stable during trypsin digestion at pH 8 but were cleaved during pepsin digestion at pH 2. The P-N bond in tabun was also acid labile, whereas the P-O bond was stable. A scheme to distinguish aging by deamination from aging by O-dealkylation was based on the acid labile P-N bond. BChE was inhibited with Sp- and Rp-tabun thiocholine nerve agent model compounds to make adducts identical to those of tabun with known stereochemistry. After aging and digestion with pepsin at pH 2, peptide FGES198AGAAS from Sp-tabun thiocholine had a mass of 902.2 m/z in negative mode, indicating that it had aged by deamination, whereas peptide FGES198AGAAS from Rp-tabun thiocholine had a mass of 874.2 m/z in negative mode, indicating that it had aged by O-dealkylation. BChE inhibited by authentic, racemic tabun yielded both 902.2 and 874.2 m/z peptides, indicating that both stereoisomers reacted with BChE and aged either by deamination or dealkylation.
ESTHER : Jiang_2013_Toxicol.Sci_132_390
PubMedSearch : Jiang_2013_Toxicol.Sci_132_390
PubMedID: 23345579

Title : This special Issue of Chemico-Biological Interactions comprises 70 manuscripts from lectures and short talks given at the 11th International Meeting on Cholinesterases. Preface -
Author(s) : Lushchekina SV , Masson P , Rosenberry TL
Ref : Chemico-Biological Interactions , 203 :1 , 2013
PubMedID: 23558087

Title : Relation between dynamics, activity and thermal stability within the cholinesterase family - Trovaslet_2013_Chem.Biol.Interact_203_14
Author(s) : Trovaslet M , Trapp M , Weik M , Nachon F , Masson P , Tehei M , Peters J
Ref : Chemico-Biological Interactions , 203 :14 , 2013
Abstract : Incoherent neutron scattering is one of the most powerful tools for studying dynamics in biological matter. Using the cold neutron backscattering spectrometer IN16 at the Institut Laue Langevin (ILL, Grenoble, France), temperature dependence of cholinesterases' dynamics (human butyrylcholinesterase from plasma: hBChE; recombinant human acetylcholinesterase: hAChE and recombinant mouse acetylcholinesterase: mAChE) was examined using elastic incoherent neutron scattering (EINS). The dynamics was characterized by the averaged atomic mean square displacement (MSD), associated with the sample flexibility at a given temperature. We found MSD values of hAChE above the dynamical transition temperature (around 200K) larger than for mAChE and hBChE, implying that hAChE is more flexible than the other ChEs. Activation energies for thermodynamical transition were extracted through the frequency window model (FWM) (Becker et al. 2004) [1] and turned out to increase from hBChE to mAChE and finally to hAChE, inversely to the MSDs relations. Between 280 and 316K, catalytic studies of these enzymes were carried out using thiocholine esters: at the same temperature, the hAChE activity was systematically higher than the mAChE or hBChE ones. Our results thus suggest a strong correlation between dynamics and activity within the ChE family. We also studied and compared the ChEs thermal inactivation kinetics. Here, no direct correlation with the dynamics was observed, thus suggesting that relations between enzyme dynamics and catalytic stability are more complex. Finally, the possible relation between flexibility and protein ability to grow in crystals is discussed.
ESTHER : Trovaslet_2013_Chem.Biol.Interact_203_14
PubMedSearch : Trovaslet_2013_Chem.Biol.Interact_203_14
PubMedID: 22940283

Title : Progress in the development of enzyme-based nerve agent bioscavengers - Nachon_2013_Chem.Biol.Interact_206_536
Author(s) : Nachon F , Brazzolotto X , Trovaslet M , Masson P
Ref : Chemico-Biological Interactions , 206 :536 , 2013
Abstract : Acetylcholinesterase is the physiological target for acute toxicity of nerve agents. Attempts to protect acetylcholinesterase from phosphylation by nerve agents, is currently achieved by reversible inhibitors that transiently mask the enzyme active site. This approach either protects only peripheral acetylcholinesterase or may cause side effects. Thus, an alternative strategy consists in scavenging nerve agents in the bloodstream before they can reach acetylcholinesterase. Pre- or post-exposure administration of bioscavengers, enzymes that neutralize and detoxify organophosphorus molecules, is one of the major developments of new medical counter-measures. These enzymes act either as stoichiometric or catalytic bioscavengers. Human butyrylcholinesterase is the leading stoichiometric bioscavenger. Current efforts are devoted to its mass production with care to pharmacokinetic properties of the final product for extended lifetime. Development of specific reactivators of phosphylated butyrylcholinesterase, or variants with spontaneous reactivation activity is also envisioned for rapid in situ regeneration of the scavenger. Human paraoxonase 1 is the leading catalytic bioscavenger under development. Research efforts focus on improving its catalytic efficiency toward the most toxic isomers of nerve agents, by means of directed evolution-based strategies. Human prolidase appears to be another promising human enzyme. Other non-human efficient enzymes like bacterial phosphotriesterases or squid diisopropylfluorophosphatase are also considered though their intrinsic immunogenic properties remain challenging for use in humans. Encapsulation, PEGylation and other modifications are possible solutions to address this problem as well as that of their limited lifetime. Finally, gene therapy for in situ generation and delivery of bioscavengers is for the far future, but its proof of concept has been established.
ESTHER : Nachon_2013_Chem.Biol.Interact_206_536
PubMedSearch : Nachon_2013_Chem.Biol.Interact_206_536
PubMedID: 23811386

Title : Polyclonal antibody to soman-tyrosine - Li_2013_Chem.Res.Toxicol_26_584
Author(s) : Li B , Duysen EG , Froment MT , Masson P , Nachon F , Jiang W , Schopfer LM , Thiele GM , Klassen LW , Cashman JR , Williams GR , Lockridge O
Ref : Chemical Research in Toxicology , 26 :584 , 2013
Abstract : Soman forms a stable, covalent bond with tyrosine 411 of human albumin, with tyrosines 257 and 593 in human transferrin, and with tyrosine in many other proteins. The pinacolyl group of soman is retained, suggesting that pinacolyl methylphosphonate bound to tyrosine could generate specific antibodies. Tyrosine in the pentapeptide RYGRK was covalently modified with soman simply by adding soman to the peptide. The phosphonylated-peptide was linked to keyhole limpet hemocyanin, and the conjugate was injected into rabbits. The polyclonal antiserum recognized soman-labeled human albumin, soman-mouse albumin, and soman human transferrin but not nonphosphonylated control proteins. The soman-labeled tyrosines in these proteins are surrounded by different amino acid sequences, suggesting that the polyclonal recognizes soman-tyrosine independent of the amino acid sequence. Antiserum obtained after 4 antigen injections over a period of 18 weeks was tested in a competition ELISA where it had an IC50 of 10(-11) M. The limit of detection on Western blots was 0.01 mug (15 picomoles) of soman-labeled albumin. In conclusion, a high-affinity, polyclonal antibody that specifically recognizes soman adducts on tyrosine in a variety of proteins has been produced. Such an antibody could be useful for identifying secondary targets of soman toxicity.
ESTHER : Li_2013_Chem.Res.Toxicol_26_584
PubMedSearch : Li_2013_Chem.Res.Toxicol_26_584
PubMedID: 23469927

Title : Strategies for the selection of catalytic antibodies against organophosphorus nerve agents - Smirnov_2013_Chem.Biol.Interact_203_196
Author(s) : Smirnov IV , Belogurov AA, Jr. , Friboulet A , Masson P , Gabibov AG , Renard PY
Ref : Chemico-Biological Interactions , 203 :196 , 2013
Abstract : Among the strategies aimed at biocompatible means for organophosphorus nerve agents neutralization, immunoglobulins have attracted attention in the 1990's and 2000's both for their ability to immobilize the toxicants, but also for their ability to be turned into enzymatically active antibodies known as catalytic antibodies or abzymes (antibodies - enzymes). We will present here a critical review of the successive strategies used for the selection of these nerve agent-hydrolyzing abzymes, based on hapten design, namely antibodies raised against a wide variety of transition state analogs, and eventually the strategies based on anti-idiotypic antibodies and reactibodies.
ESTHER : Smirnov_2013_Chem.Biol.Interact_203_196
PubMedSearch : Smirnov_2013_Chem.Biol.Interact_203_196
PubMedID: 23123255

Title : Matrix-assisted laser desorption\/ionization time-of-flight mass spectrometry of titanium oxide-enriched peptides for detection of aged organophosphorus adducts on human butyrylcholinesterase - Jiang_2013_Anal.Biochem_439_132
Author(s) : Jiang W , Murashko EA , Dubrovskii YA , Podolskaya EP , Babakov VN , Mikler J , Nachon F , Masson P , Schopfer LM , Lockridge O
Ref : Analytical Biochemistry , 439 :132 , 2013
Abstract : Exposure to nerve agents or organophosphorus (OP) pesticides can have life-threatening effects. Human plasma butyrylcholinesterase (BChE) inactivates these poisons by binding them to Ser198. After hours or days, these OP adducts acquire a negative charge by dealkylation in a process called aging. Our goal was to develop a method for enriching the aged adduct to facilitate detection of exposure. Human BChE inhibited by OP toxicants was incubated for 4days to 6years. Peptides produced by digestion with pepsin were enriched by binding to titanium oxide (TiO2) and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. It was found that with two exceptions, all aged OP adducts in peptide FGES198AGAAS were enriched by binding to Titansphere tips. Cresyl saligenin phosphate yielded two types of aged adduct, cresylphosphate and phosphate, but only the phosphate adduct bound to Titansphere. The nerve agent VR yielded no aged adduct, supporting crystal structure findings that the VR adduct on BChE does not age. The irreversible nature of aged OP adducts was demonstrated by the finding that after 6years at room temperature in sterile pH 7.0 buffer, the adducts were still detectable. It was concluded that TiO2 microcolumns can be used to enrich aged OP-modified BChE peptide.
ESTHER : Jiang_2013_Anal.Biochem_439_132
PubMedSearch : Jiang_2013_Anal.Biochem_439_132
PubMedID: 23624322

Title : Research on cholinesterases in the Soviet Union and Russia: a historical perspective - Rozengart_2013_Chem.Biol.Interact_203_3
Author(s) : Rozengart EV , Basova NE , Moralev SN , Lushchekina SV , Masson P , Varfolomeev SD
Ref : Chemico-Biological Interactions , 203 :3 , 2013
Abstract : Research on cholinesterases and effects of their inhibition in the USSR and Russia since 1930-1940s till present is exposed in historical aspects. The first physiological and toxicological effects of cholinesterase inhibition were reported by Alexander Ginetsinsky during World War II, when academic institutions were evacuated from Leningrad to Kazan. The main scientific schools that initiated research on chemistry, enzymology and physiology of cholinesterases and their inhibitors were leaded by Alexandr and Boris Arbuzovs, Victor Rozengart, Viktor Yakovlev, Michael Michelson, Martin Kabachnik, Mikhail Voronkov, Ivan Knunyants, Alexandr Bretskin and others. They investigated the main physiological effects of cholinesterase inhibitors, and analyzed the catalytic mechanisms of cholinesterases and related enzymes. Their contributions are landmarks in the history of cholinesterase research. At the present time revival of research on cholinesterases in different universities and institutes is vivid, in particular at the Moscow State University, research institutes of Russian Academy of Sciences and Kazan Scientific Center.
ESTHER : Rozengart_2013_Chem.Biol.Interact_203_3
PubMedSearch : Rozengart_2013_Chem.Biol.Interact_203_3
PubMedID: 23485591

Title : Recombinant human butyrylcholinesterase as a new-age bioscavenger drug: development of the expression system - Ilyushin_2013_Acta.Naturae_5_73
Author(s) : Ilyushin DG , Haertley OM , Bobik TV , Shamborant OG , Surina EA , Knorre VD , Masson P , Smirnov IV , Gabibov AG , Ponomarenko NA
Ref : Acta Naturae , 5 :73 , 2013
Abstract : Butyrylcholinesterase (BChE) is a serine hydrolase (EC 3.1.1.8) which can be found in most animal tissues. This enzyme has a broad spectrum of efficacy against organophosphorus compounds, which makes it a prime candidate for the role of stoichiometric bioscavenger. Development of a new-age DNA-encoded bioscavenger is a vival task. Several transgenic expression systems of human BChE were developed over the past 20 years; however, none of them has been shown to make economic sense or has been approved for administration to humans. In this study, a CHO-based expression system was redesigned, resulting in a significant increase in the production level of functional recombinant human butyrylcholinesterase as compared to the hitherto existing systems. The recombinant enzyme was characterized with Elman and ELISA methods.
ESTHER : Ilyushin_2013_Acta.Naturae_5_73
PubMedSearch : Ilyushin_2013_Acta.Naturae_5_73
PubMedID: 23556132

Title : Inhibition pathways of the potent organophosphate CBDP with cholinesterases revealed by X-ray crystallographic snapshots and mass spectrometry - Carletti_2013_Chem.Res.Toxicol_26_280
Author(s) : Carletti E , Colletier JP , Schopfer LM , Santoni G , Masson P , Lockridge O , Nachon F , Weik M
Ref : Chemical Research in Toxicology , 26 :280 , 2013
Abstract : Tri-o-cresyl-phosphate (TOCP) is a common additive in jet engine lubricants and hydraulic fluids suspected to have a role in aerotoxic syndrome in humans. TOCP is metabolized to cresyl saligenin phosphate (CBDP), a potent irreversible inhibitor of butyrylcholinesterase (BChE), a natural bioscavenger present in the bloodstream, and acetylcholinesterase (AChE), the off-switch at cholinergic synapses. Mechanistic details of cholinesterase (ChE) inhibition have, however, remained elusive. Also, the inhibition of AChE by CBDP is unexpected, from a structural standpoint, i.e., considering the narrowness of AChE active site and the bulkiness of CBDP. In the following, we report on kinetic X-ray crystallography experiments that provided 2.7-3.3 A snapshots of the reaction of CBDP with mouse AChE and human BChE. The series of crystallographic snapshots reveals that AChE and BChE react with the opposite enantiomers and that an induced-fit rearrangement of Phe297 enlarges the active site of AChE upon CBDP binding. Mass spectrometry analysis of aging in either H(2)(16)O or H(2)(18)O furthermore allowed us to identify the inhibition steps, in which water molecules are involved, thus providing insights into the mechanistic details of inhibition. X-ray crystallography and mass spectrometry show the formation of an aged end product formed in both AChE and BChE that cannot be reactivated by current oxime-based therapeutics. Our study thus shows that only prophylactic and symptomatic treatments are viable to counter the inhibition of AChE and BChE by CBDP.
ESTHER : Carletti_2013_Chem.Res.Toxicol_26_280
PubMedSearch : Carletti_2013_Chem.Res.Toxicol_26_280
PubMedID: 23339663
Gene_locus related to this paper: human-BCHE , mouse-ACHE

Title : Effects of hydrostatic pressure on the quaternary structure and enzymatic activity of a large peptidase complex from Pyrococcus horikoshii - Rosenbaum_2012_Arch.Biochem.Biophys_517_104
Author(s) : Rosenbaum E , Gabel F , Dura MA , Finet S , Clery-Barraud C , Masson P , Franzetti B
Ref : Archives of Biochemistry & Biophysics , 517 :104 , 2012
Abstract : While molecular adaptation to high temperature has been extensively studied, the effect of hydrostatic pressure on protein structure and enzymatic activity is still poorly understood. We have studied the influence of pressure on both the quaternary structure and enzymatic activity of the dodecameric TET3 peptidase from Pyrococcus horikoshii. Small angle X-ray scattering (SAXS) revealed a high robustness of the oligomer under high pressure of up to 300 MPa at 25 degrees C as well as at 90 degrees C. The enzymatic activity of TET3 was enhanced by pressure up to 180 MPa. From the pressure behavior of the different rate-constants we have determined the volume changes associated with substrate binding and catalysis. Based on these results we propose that a change in the rate-limiting step occurs around 180 MPa.
ESTHER : Rosenbaum_2012_Arch.Biochem.Biophys_517_104
PubMedSearch : Rosenbaum_2012_Arch.Biochem.Biophys_517_104
PubMedID: 21896270

Title : Activity and molecular dynamics relationship within the family of human cholinesterases - Peters_2012_Phys.Chem.Chem.Phys_14_6764
Author(s) : Peters J , Trovaslet M , Trapp M , Nachon F , Hill F , Royer E , Gabel F , van Eijck L , Masson P , Tehei M
Ref : Phys Chem Chem Phys , 14 :6764 , 2012
Abstract : The temperature dependence of the dynamics of recombinant human acetylcholinesterase (hAChE) and plasma human butyrylcholinesterase (hBChE) is examined using elastic incoherent neutron scattering. These two enzymes belong to the same family and present 50% amino acid sequence identity. However, significantly higher flexibility and catalytic activity of hAChE when compared to the ones of hBChE are measured. At the same time, the average height of the potential barrier to the motions is increased in the hBChE, e.g. more thermal energy is needed to cross it in the latter case, which might be the origin of the increase in activation energy and the reduction in the catalytic rate of hBChE observed experimentally. These results suggest that the motions on the picosecond timescale may act as a lubricant for those associated with activity occurring on a slower millisecond timescale.
ESTHER : Peters_2012_Phys.Chem.Chem.Phys_14_6764
PubMedSearch : Peters_2012_Phys.Chem.Chem.Phys_14_6764
PubMedID: 22395795

Title : Time-dependent kinetic complexities in cholinesterase-catalyzed reactions - Masson_2012_Biochemistry.(Mosc)_77_1147
Author(s) : Masson P
Ref : Biochemistry (Mosc) , 77 :1147 , 2012
Abstract : Cholinesterases ChEs display a hysteretic behavior with certain substrates and inhibitors. Kinetic cooperativity in hysteresis of ChE-catalyzed reactions is characterized by a lag or burst phase in the approach to steady state. With some substrates damped oscillations are shown to superimpose on hysteretic lags. These time dependent peculiarities are observed for both butyrylcholinesterase and acetylcholinesterase from different sources Hysteresis in ChE-catalyzed reactions can be interpreted in terms of slow transitions between two enzyme conformers E and E . Substrate can bind to E and/or E both Michaelian complexes ES and Epsilon'S can be catalytically competent or only one of them can make products. The formal reaction pathway depends on both the chemical structure of the substrate and the type of enzyme. In particular damped oscillations develop when substrate exists in different slowly interconvertible conformational and/or micellar forms of which only the minor form is capable of binding and reacting with the enzyme. Biphasic pseudo-first-order progressive inhibition of ChEs by certain carbamates and organophosphates also fits with a slow equilibrium between two reactive enzyme forms. Hysteresis can be modulated by medium parameters, pH, chaotropic and kosmotropic salts, organic solvents, temperature, osmotic pressure and hydrostatic pressure. These studies showed that water structure plays a role in hysteretic behavior of ChEs. Attempts to provide a molecular mechanism for ChE hysteresis from mutagenesis studies or crystallographic studies failed so far. In fact several lines of evidence suggest that hysteresis is controlled by the conformation of His438 a key residue in the catalytic triad of cholinesterases. Induction time may depend on the probability of His438 to adopt the operative conformation in the catalytic triad. The functional significance of ChE hysteresis is puzzling. However the accepted view that proteins are in equilibrium between preexisting functional and non-functional conformers and that binding of a ligand to the functional form shifts equilibrium towards the functional conformation suggests that slow equilibrium between two conformational states of these enzymes may have a regulatory function in damping out the response to certain ligands and irreversible inhibitors. This is particularly true for immobilized membrane bound enzymes where the local substrate and/or inhibitor concentrations depend on influx in crowded organellar systems e.g cholinergic synaptic clefts. Therefore physiological or toxicological relevance of the hysteretic behavior and damped oscillations in ChE-catalyzed reactions and inhibition cannot be ruled out.
ESTHER : Masson_2012_Biochemistry.(Mosc)_77_1147
PubMedSearch : Masson_2012_Biochemistry.(Mosc)_77_1147
PubMedID: 23157295

Title : Energy landscapes of human acetylcholinesterase and its huperzine a-inhibited counterpart - Trapp_2012_J.Phys.Chem.B_116_14744
Author(s) : Trapp M , Trovaslet M , Nachon F , Koza MM , van Eijck L , Hill F , Weik M , Masson P , Tehei M , Peters J
Ref : J Phys Chem B , 116 :14744 , 2012
Abstract : Enzymes are animated by a hierarchy of motions occurring on time scales that span more than 15 orders of magnitude from femtoseconds (10(-15) s) to several minutes. As a consequence, an enzyme is characterized by a large number of conformations, so-called conformational substates that interconvert via molecular motions. The energy landscapes of these macromolecules are very complex, and many conformations are separated by only small energy barriers. Movements at this level are fast thermal atomic motions occurring on a time scale between 10(-7) and 10(-12) s, which are experimentally accessible by incoherent neutron scattering techniques. They correspond to local fluctuations within the molecule and are believed to act as coupling links for larger, conformational changes. Several questions related to this hierarchy of motions are a matter of very active research: which of the motions are involved in the biological functions of the macromolecule and are motions of different energy (and thus time) scale correlated? How does the distribution of motions change when an enzyme is inhibited? We report here on investigations of the enzyme human acetylcholinesterase, unliganded and in complex with the noncovalent inhibitor Huperzine A, by incoherent neutron scattering. Different time scales are explored to shed light on the interplay of enzyme activity, dynamics, and inhibition. Surprisingly the average molecular dynamics do not seem to be altered by the presence of the inhibitor used in this study within the considered time scales. The activation energy for the free and the inhibited form of the enzyme is moreover found to be almost identical despite changes of interactions inside the gorge, which leads to the active site of the enzyme.
ESTHER : Trapp_2012_J.Phys.Chem.B_116_14744
PubMedSearch : Trapp_2012_J.Phys.Chem.B_116_14744
PubMedID: 23186408

Title : Differential sensitivity of plasma carboxylesterase-null mice to parathion, chlorpyrifos and chlorpyrifos oxon, but not to diazinon, dichlorvos, diisopropylfluorophosphate, cresyl saligenin phosphate, cyclosarin thiocholine, tabun thiocholine, and carbofuran - Duysen_2012_Chem.Biol.Interact_195_189
Author(s) : Duysen EG , Cashman JR , Schopfer LM , Nachon F , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 195 :189 , 2012
Abstract : Mouse blood contains four esterases that detoxify organophosphorus compounds: carboxylesterase, butyrylcholinesterase, acetylcholinesterase, and paraoxonase-1. In contrast human blood contains the latter three enzymes but not carboxylesterase. Organophosphorus compound toxicity is due to inhibition of acetylcholinesterase. Symptoms of intoxication appear after approximately 50% of the acetylcholinesterase is inhibited. However, complete inhibition of carboxylesterase and butyrylcholinesterase has no known effect on an animal's well being. Paraoxonase hydrolyzes organophosphorus compounds and is not inhibited by them. Our goal was to determine the effect of plasma carboxylesterase deficiency on response to sublethal doses of 10 organophosphorus toxicants and one carbamate pesticide. Homozygous plasma carboxylesterase deficient ES1(-/-) mice and wild-type littermates were observed for toxic signs and changes in body temperature after treatment with a single sublethal dose of toxicant. Inhibition of plasma acetylcholinesterase, butyrylcholinesterase, and plasma carboxylesterase was measured. It was found that wild-type mice were protected from the toxicity of 12.5mg/kg parathion applied subcutaneously. However, both genotypes responded similarly to paraoxon, cresyl saligenin phosphate, diisopropylfluorophosphate, diazinon, dichlorvos, cyclosarin thiocholine, tabun thiocholine, and carbofuran. An unexpected result was the finding that transdermal application of chlorpyrifos at 100mg/kg and chlorpyrifos oxon at 14mg/kg was lethal to wild-type but not to ES1(-/-) mice, showing that with this organochlorine, the presence of carboxylesterase was harmful rather than protective. It was concluded that carboxylesterase in mouse plasma protects from high toxicity agents, but the amount of carboxylesterase in plasma is too low to protect from low toxicity compounds that require high doses to inhibit acetylcholinesterase.
ESTHER : Duysen_2012_Chem.Biol.Interact_195_189
PubMedSearch : Duysen_2012_Chem.Biol.Interact_195_189
PubMedID: 22209767

Title : Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics - Smirnov_2011_Proc.Natl.Acad.Sci.U.S.A_108_15954
Author(s) : Smirnov IV , Carletti E , Kurkova I , Nachon F , Nicolet Y , Mitkevich VA , Debat H , Avalle B , Belogurov AA, Jr. , Kuznetsov N , Reshetnyak A , Masson P , Tonevitsky AG , Ponomarenko NA , Makarov AA , Friboulet A , Tramontano A , Gabibov AG
Ref : Proc Natl Acad Sci U S A , 108 :15954 , 2011
Abstract : Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-A-deep two-chamber cavity at the interface of variable light (V(L)) and variable heavy (V(H)) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.
ESTHER : Smirnov_2011_Proc.Natl.Acad.Sci.U.S.A_108_15954
PubMedSearch : Smirnov_2011_Proc.Natl.Acad.Sci.U.S.A_108_15954
PubMedID: 21896761

Title : Evolution of and perspectives on therapeutic approaches to nerve agent poisoning - Masson_2011_Toxicol.Lett_206_5
Author(s) : Masson P
Ref : Toxicol Lett , 206 :5 , 2011
Abstract : After more than 70 years of considerable efforts, research on medical defense against nerve agents has come to a standstill. Major progress in medical countermeasures was achieved between the 50s and 70s with the development of anticholinergic drugs and carbamate-based pretreatment, the introduction of pyridinium oximes as antidotes, and benzodiazepines in emergency treatments. These drugs ensure good protection of the peripheral nervous system and mitigate the acute effects of exposure to lethal doses of nerve agents. However, pyridostigmine and cholinesterase reactivators currently used in the armed forces do not protect/reactivate central acetylcholinesterases. Moreover, other drugs used are not sufficiently effective in protecting the central nervous system against seizures, irreversible brain damages and long-term sequelae of nerve agent poisoning.New developments of medical counter-measures focus on: (a) detoxification of organophosphorus molecules before they react with acetylcholinesterase and other physiological targets by administration of stoichiometric or catalytic scavengers; (b) protection and reactivation of central acetylcholinesterases, and (c) improvement of neuroprotection following delayed therapy.Future developments will aim at treatment of acute and long-term effects of low level exposure to nerve agents, research on alternative routes for optimizing drug delivery, and therapies. Though gene therapy for in situ generation of bioscavengers, and cell therapy based on neural progenitor engraftment for neuronal regeneration have been successfully explored, more studies are needed before practical medical applications can be made of these new approaches.
ESTHER : Masson_2011_Toxicol.Lett_206_5
PubMedSearch : Masson_2011_Toxicol.Lett_206_5
PubMedID: 21524695

Title : Organophosphate hydrolases as catalytic bioscavengers of organophosphorus nerve agents - Trovaslet-Leroy_2011_Toxicol.Lett_206_14
Author(s) : Trovaslet-Leroy M , Musilova L , Renault F , Brazzolotto X , Misik J , Novotny L , Froment MT , Gillon E , Loiodice M , Verdier L , Masson P , Rochu D , Jun D , Nachon F
Ref : Toxicol Lett , 206 :14 , 2011
Abstract : Bioscavengers are molecules able to neutralize neurotoxic organophosphorus compounds (OP) before they can reach their biological target. Human butyrylcholinesterase (hBChE) is a natural bioscavenger each molecule of enzyme neutralizing one molecule of OP. The amount of natural enzyme is insufficient to achieve good protection. Thus, different strategies have been envisioned. The most straightforward consists in injecting a large dose of highly purified natural hBChE to increase the amount of bioscavenger in the bloodstream. This proved to be successful for protection against lethal doses of soman and VX but remains expensive. An improved strategy is to regenerate prophylactic cholinesterases (ChE) by administration of reactivators after exposure. But broad-spectrum efficient reactivators are still lacking, especially for inhibited hBChE. Cholinesterase mutants capable of reactivating spontaneously are another option. The G117H hBChE mutant has been a prototype. We present here the Y124H/Y72D mutant of human acetylcholinesterase; its spontaneous reactivation rate after V-agent inhibition is increased up to 110 fold. Catalytic bioscavengers, enzymes capable of hydrolyzing OP, present the best alternative. Mesophilic bacterial phosphotriesterase (PTE) is a candidate with good catalytic efficiency. Its enantioselectivity has been enhanced against the most potent OP isomers by rational design. We show that PEGylation of this enzyme improves its mean residence time in the rat blood stream 24-fold and its bioavailability 120-fold. Immunogenic issues remain to be solved. Human paraoxonase 1 (hPON1) is another promising candidate. However, its main drawback is that its phosphotriesterase activity is highly dependent on its environment. Recent progress has been made using a mammalian chimera of PON1, but we provide here additional data showing that this chimera is biochemically different from hPON1. Besides, the chimera is expected to suffer from immunogenic issues. Thus, we stress that interest for hPON1 must not fade away, and in particular, the 3D structure of the hPON1 eventually in complex with OP has to be solved.
ESTHER : Trovaslet-Leroy_2011_Toxicol.Lett_206_14
PubMedSearch : Trovaslet-Leroy_2011_Toxicol.Lett_206_14
PubMedID: 21683774

Title : X-ray crystallographic snapshots of reaction intermediates in the G117H mutant of human butyrylcholinesterase, a nerve agent target engineered into a catalytic bioscavenger - Nachon_2011_Biochem.J_434_73
Author(s) : Nachon F , Carletti E , Wandhammer M , Nicolet Y , Schopfer LM , Masson P , Lockridge O
Ref : Biochemical Journal , 434 :73 , 2011
Abstract : OPs (organophosphylates) exert their acute toxicity through inhibition of acetylcholinesterase, by phosphylation of the catalytic serine residue. Engineering of human butyrylcholinesterase, by substitution of a histidine residue for the glycine residue at position 117, led to the creation of OP hydrolase activity. However, the lack of structural information and poor understanding of the hydrolytic mechanism of the G117H mutant has hampered further improvements in the catalytic activity. We have solved the crystallographic structure of the G117H mutant with a variety of ligands in its active site. A sulfate anion bound to the active site suggested the positioning for an OP prior to phosphylation. A fluoride anion was found in the active site when NaF was added to the crystallization buffer. In the fluoride complex, the imidazole ring from the His117 residue was substantially shifted, adopting a relaxed conformation probably close to that of the unliganded mutant enzyme. Additional X-ray structures were obtained from the transient covalent adducts formed upon reaction of the G117H mutant with the OPs echothiophate and VX [ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl](methyl)phosphinate]. The position of the His117 residue shifted in response to the introduction of these adducts, overlaying the phosphylserine residue. These structural data suggest that the dephosphylation mechanism involves either a substantial conformational change of the His117 residue or an adjacent nucleophilic substitution by water.
ESTHER : Nachon_2011_Biochem.J_434_73
PubMedSearch : Nachon_2011_Biochem.J_434_73
PubMedID: 21091433
Gene_locus related to this paper: human-BCHE

Title : Reaction of cresyl saligenin phosphate, the organophosphorus agent implicated in aerotoxic syndrome, with human cholinesterases: mechanistic studies employing kinetics, mass spectrometry, and X-ray structure analysis - Carletti_2011_Chem.Res.Toxicol_24_797
Author(s) : Carletti E , Schopfer LM , Colletier JP , Froment MT , Nachon F , Weik M , Lockridge O , Masson P
Ref : Chemical Research in Toxicology , 24 :797 , 2011
Abstract : Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate (TCP), an antiwear additive in jet engine lubricants and hydraulic fluid. CBDP (2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one) is the toxic metabolite of triortho-cresylphosphate, a component of TCP. Human butyrylcholinesterase (BChE; EC 3.1.1.8) and human acetylcholinesterase (AChE; EC 3.1.1.7) are irreversibly inhibited by CBDP. The bimolecular rate constants of inhibition (k(i)), determined under pseudo-first-order conditions, displayed a biphasic time course of inhibition with k(i) of 1.6 x 10(8) M(-1) min(-1) and 2.7 x 10(7) M(-1) min(-1) for E and E' forms of BChE. The inhibition constants for AChE were 1 to 2 orders of magnitude slower than those for BChE. CBDP-phosphorylated cholinesterases are nonreactivatable due to ultra fast aging. Mass spectrometry analysis showed an initial BChE adduct with an added mass of 170 Da from cresylphosphate, followed by dealkylation to a structure with an added mass of 80 Da. Mass spectrometry in (18)O-water showed that (18)O was incorporated only during the final aging step to form phospho-serine as the final aged BChE adduct. The crystal structure of CBDP-inhibited BChE confirmed that the phosphate adduct is the ultimate aging product. CBDP is the first organophosphorus agent that leads to a fully dealkylated phospho-serine BChE adduct.
ESTHER : Carletti_2011_Chem.Res.Toxicol_24_797
PubMedSearch : Carletti_2011_Chem.Res.Toxicol_24_797
PubMedID: 21438623
Gene_locus related to this paper: human-BCHE

Title : Structural study of the complex stereoselectivity of human butyrylcholinesterase for the neurotoxic V-agents - Wandhammer_2011_J.Biol.Chem_286_16783
Author(s) : Wandhammer M , Carletti E , van der Schans M , Gillon E , Nicolet Y , Masson P , Goeldner M , Noort D , Nachon F
Ref : Journal of Biological Chemistry , 286 :16783 , 2011
Abstract : Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the P(S) adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VX(R)-(+) and VX(S)-(-) solutions lead to the formation of the P(S) and P(R) adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VX(R)-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VX(S)-(-). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with P(R) enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the P(S) enantiomer and ages.
ESTHER : Wandhammer_2011_J.Biol.Chem_286_16783
PubMedSearch : Wandhammer_2011_J.Biol.Chem_286_16783
PubMedID: 21454498
Gene_locus related to this paper: human-BCHE

Title : Exposure to tri-o-cresyl phosphate detected in jet airplane passengers - Liyasova_2011_Toxicol.Appl.Pharmacol_256_337
Author(s) : Liyasova M , Li B , Schopfer LM , Nachon F , Masson P , Furlong CE , Lockridge O
Ref : Toxicol Appl Pharmacol , 256 :337 , 2011
Abstract : The aircraft cabin and flight deck ventilation are supplied from partially compressed unfiltered bleed air directly from the engine. Worn or defective engine seals can result in the release of engine oil into the cabin air supply. Aircrew and passengers have complained of illness following such "fume events". Adverse health effects are hypothesized to result from exposure to tricresyl phosphate mixed esters, a chemical added to jet engine oil and hydraulic fluid for its anti-wear properties. Our goal was to develop a laboratory test for exposure to tricresyl phosphate. The assay was based on the fact that the active-site serine of butyrylcholinesterase reacts with the active metabolite of tri-o-cresyl phosphate, cresyl saligenin phosphate, to make a stable phosphorylated adduct with an added mass of 80 Da. No other organophosphorus agent makes this adduct in vivo on butyrylcholinesterase. Blood samples from jet airplane passengers were obtained 24-48 h after completing a flight. Butyrylcholinesterase was partially purified from 25 ml serum or plasma, digested with pepsin, enriched for phosphorylated peptides by binding to titanium oxide, and analyzed by mass spectrometry. Of 12 jet airplane passengers tested, 6 were positive for exposure to tri-o-cresyl phosphate that is, they had detectable amounts of the phosphorylated peptide FGEpSAGAAS. The level of exposure was very low. No more than 0.05 to 3% of plasma butyrylcholinesterase was modified. None of the subjects had toxic symptoms. Four of the positive subjects were retested 3 to 7 months following their last airplane trip and were found to be negative for phosphorylated butyrylcholinesterase. In conclusion, this is the first report of an assay that detects exposure to tri-o-cresyl phosphate in jet airplane travelers.
ESTHER : Liyasova_2011_Toxicol.Appl.Pharmacol_256_337
PubMedSearch : Liyasova_2011_Toxicol.Appl.Pharmacol_256_337
PubMedID: 21723309

Title : Aging mechanism of butyrylcholinesterase inhibited by an N-methyl analogue of tabun: implications of the trigonal-bipyramidal transition state rearrangement for the phosphylation or reactivation of cholinesterases - Nachon_2010_Chem.Biol.Interact_187_44
Author(s) : Nachon F , Carletti E , Worek F , Masson P
Ref : Chemico-Biological Interactions , 187 :44 , 2010
Abstract : Cholinesterases are the main target of organophosphorus nerve agents (OPs). Their inhibition results in cholinergic syndrome and death. The enzymes are inhibited by phosphylation of the catalytic serine enzyme, but can be reactivated by oximes to some extent. However, phosphylated cholinesterases undergo a side reaction that progressively prevents their reactivatability. This unimolecular reaction, termed "aging", has been investigated for decades. It was shown that most OP-ChE conjugates aged by O-dealkylation of an alkoxy substituent of the phosphorus atom, a mechanism involving the stabilization of a transient carbocation. In this paper we present structural data supporting a substitution-based mechanism for aging of the huBChE conjugate of an N-mono-methyl analogue of tabun. This mechanism involves an adjacent nucleophilic attack followed by Berry pseudorotation. A similar adjacent attack and subsequent rearrangement of the transition state have been recently proposed for tabun phosphylation of AChE. We suggest that a similar mechanism is also possible for oxime reactivation of phosphylated cholinesterases. This opens new perspectives in terms of reactivator design.
ESTHER : Nachon_2010_Chem.Biol.Interact_187_44
PubMedSearch : Nachon_2010_Chem.Biol.Interact_187_44
PubMedID: 20381476

Title : Mass spectral characterization of organophosphate-labeled, tyrosine-containing peptides: characteristic mass fragments and a new binding motif for organophosphates - Schopfer_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1297
Author(s) : Schopfer LM , Grigoryan H , Li B , Nachon F , Masson P , Lockridge O
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 878 :1297 , 2010
Abstract : We have identified organophosphorus agent (OP)-tyrosine adducts on 12 different proteins labeled with six different OP. Labeling was achieved by treating pure proteins with up to 40-fold molar excess of OP at pH 8-8.6. OP-treated proteins were digested with trypsin, and peptides were separated by HPLC. Fragmentation patterns for 100 OP-peptides labeled on tyrosine were determined in the mass spectrometer. The goals of the present work were (1) to determine the common features of the OP-reactive tyrosines, and (2) to describe non-sequence MSMS fragments characteristic of OP-tyrosine peptides. Characteristic ions at 272 and 244 amu for tyrosine-OP immonium ions were nearly always present in the MSMS spectrum of peptides labeled on tyrosine by chlorpyrifos-oxon. Characteristic fragments also appeared from the parent ions that had been labeled with diisopropylfluorophosphate (216 amu), sarin (214 amu), soman (214 amu) or FP-biotin (227, 312, 329, 691 and 708 amu). In contrast to OP-reactive serines, which lie in the consensus sequence GXSXG, the OP-reactive tyrosines have no consensus sequence. Their common feature is the presence of nearby positively charged residues that activate the phenolic hydroxyl group. The significance of these findings is the recognition of a new binding motif for OP to proteins that have no active site serine. Modified peptides are difficult to find when the OP bears no radiolabel and no tag. The characteristic MSMS fragment ions are valuable because they are identifiers for OP-tyrosine, independent of the peptide.
ESTHER : Schopfer_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1297
PubMedSearch : Schopfer_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1297
PubMedID: 19762289

Title : Accumulation of tetrahedral intermediates in cholinesterase catalysis: a secondary isotope effect study - Tormos_2010_J.Am.Chem.Soc_132_17751
Author(s) : Tormos JR , Wiley KL , Wang Y , Fournier D , Masson P , Nachon F , Quinn DM
Ref : Journal of the American Chemical Society , 132 :17751 , 2010
Abstract : In a previous communication, kinetic beta-deuterium secondary isotope effects were reported that support a mechanism for substrate-activated turnover of acetylthiocholine by human butyrylcholinesterase (BuChE) wherein the accumulating reactant state is a tetrahedral intermediate ( Tormos , J. R. ; et al. J. Am. Chem. Soc. 2005 , 127 , 14538 - 14539 ). In this contribution additional isotope effect experiments are described with acetyl-labeled acetylthiocholines (CL(3)COSCH(2)CH(2)N(+)Me(3); L = H or D) that also support accumulation of the tetrahedral intermediate in Drosophila melanogaster acetylcholinesterase (DmAChE) catalysis. In contrast to the aforementioned BuChE-catalyzed reaction, for this reaction the dependence of initial rates on substrate concentration is marked by pronounced substrate inhibition at high substrate concentrations. Moreover, kinetic beta-deuterium secondary isotope effects for turnover of acetylthiocholine depended on substrate concentration, and gave the following: (D3)k(cat)/K(m) = 0.95 +/- 0.03, (D3)k(cat) = 1.12 +/- 0.02 and (D3)betak(cat) = 0.97 +/- 0.04. The inverse isotope effect on k(cat)/K(m) is consistent with conversion of the sp(2)-hybridized substrate carbonyl in the E + A reactant state into a quasi-tetrahedral transition state in the acylation stage of catalysis, whereas the markedly normal isotope effect on k(cat) is consistent with hybridization change from sp(3) toward sp(2) as the reactant state for deacylation is converted into the subsequent transition state. Transition states for Drosophila melanogaster AChE-catalyzed hydrolysis of acetylthiocholine were further characterized by measuring solvent isotope effects and determining proton inventories. These experiments indicated that the transition state for rate-determining decomposition of the tetrahedral intermediate is stabilized by multiple protonic interactions. Finally, a simple model is proposed for the contribution that tetrahedral intermediate stabilization provides to the catalytic power of acetylcholinesterase.
ESTHER : Tormos_2010_J.Am.Chem.Soc_132_17751
PubMedSearch : Tormos_2010_J.Am.Chem.Soc_132_17751
PubMedID: 21105647

Title : Dichlorvos, chlorpyrifos oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdose - Li_2010_J.Appl.Toxicol_30_559
Author(s) : Li B , Ricordel I , Schopfer LM , Baud F , Megarbane B , Masson P , Lockridge O
Ref : J Appl Toxicol , 30 :559 , 2010
Abstract : The goal of this study was to develop a method to detect pesticide adducts in tryptic digests of butyrylcholinesterase in human plasma from patients poisoned by pesticides. Adducts to butyrylcholinesterase in human serum may serve as biomarkers of pesticide exposure because organophosphorus and carbamate pesticides make a covalent bond with the active site serine of butyrylcholinesterase. Serum samples from five attempted suicides (with dichlorvos, Aldicarb, Baygon and an unknown pesticide) and from one patient who accidentally inhaled dichlorvos were analyzed. Butyrylcholinesterase was purified from 2 ml serum by ion exchange chromatography at pH 4, followed by procainamide affinity chromatography at pH 7. The purified butyrylcholinesterase was denatured, digested with trypsin and the modified peptide isolated by HPLC. The purified peptide was analyzed by multiple reaction monitoring in a QTRAP 4000 mass spectrometer. This method successfully identified the pesticide-adducted butyrylcholinesterase peptide in four patients whose butyrylcholinesterase was inhibited 60-84%, but not in two patients whose inhibition levels were 8 and 22%. It is expected that low inhibition levels will require analysis of larger serum plasma volumes. In conclusion, a mass spectrometry method for identification of exposure to live toxic pesticides has been developed, based on identification of pesticide adducts on the active site serine of human butyrylcholinesterase.
ESTHER : Li_2010_J.Appl.Toxicol_30_559
PubMedSearch : Li_2010_J.Appl.Toxicol_30_559
PubMedID: 20809544

Title : Detection of adduct on tyrosine 411 of albumin in humans poisoned by dichlorvos - Li_2010_Toxicol.Sci_116_23
Author(s) : Li B , Ricordel I , Schopfer LM , Baud F , Megarbane B , Nachon F , Masson P , Lockridge O
Ref : Toxicol Sci , 116 :23 , 2010
Abstract : Studies in mice and guinea pigs have shown that albumin is a new biomarker of organophosphorus toxicant (OP) and nerve agent exposure. Our goal was to determine whether OP-labeled albumin could be detected in the blood of humans exposed to OP. Blood from four OP-exposed patients was prepared for mass spectrometry analysis by digesting 0.010 ml of serum with pepsin and purifying the labeled albumin peptide by offline high performance liquid chromatography. Dimethoxyphosphate-labeled tyrosine 411 was identified in albumin peptides VRY(411)TKKVPQVSTPTL and LVRY(411)TKKVPQVSTPTL from two patients who had attempted suicide with dichlorvos. The butyrylcholinesterase activity in these serum samples was inhibited 80%. A third patient whose serum BChE activity was inhibited 8% by accidental inhalation of dichlorvos had undetectable levels of adduct on albumin. A fourth patient whose BChE activity was inhibited 60% by exposure to chlorpyrifos had no detectable adduct on albumin. This is the first report to demonstrate the presence of OP-labeled albumin in human patients. It is concluded that tyrosine 411 of human albumin is covalently modified in the serum of humans poisoned by dichlorvos and that the modification is detectable by mass spectrometry. The special reactivity of tyrosine 411 with OP suggests that other proteins may also be modified on tyrosine. Identification of other OP-modified proteins may lead to an understanding of neurotoxic symptoms that appear long after the initial OP exposure.
ESTHER : Li_2010_Toxicol.Sci_116_23
PubMedSearch : Li_2010_Toxicol.Sci_116_23
PubMedID: 20395308

Title : Integrative analytical approach by capillary electrophoresis and kinetics under high pressure optimized for deciphering intrinsic and extrinsic cofactors that modulate activity and stability of human paraoxonase (PON1) - Renault_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1346
Author(s) : Renault F , Carus T , Clery-Barraud C , Elias M , Chabriere E , Masson P , Rochu D
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 878 :1346 , 2010
Abstract : Paraoxonase (PON1) is working in vivo in a particular dynamic environment including HDL particles and associated molecules. To decipher the respective and/or concomitant role of the different cofactors involved in this molecular organization, an approach using multiple experimental techniques based on capillary electrophoresis and classical kinetics or kinetics under high pressure was implemented. The effects of calcium and phosphate as protein or plasma cofactor, of human phosphate binding protein (HPBP) as enzyme chaperone, and of a PON1 inhibitor as an active site stabilizer, on the catalytic activities and functional oligomerization of PON1 were scrutinized. PON1 displays two distinct catalytic behaviors, one against esters and lactones, the other against organophosphorus compounds; its functional states and catalytic activities against these substrates are differently modulated by the molecular environment; PON1 exists under several active multimeric forms; the binding of HPBP amends the size of the oligomeric states and exerts a stabilizing effect on the activities of PON1; PON1 functional properties are modulated by HPBP, calcium and phosphate. This integrative approach using several optimized analytical techniques allowed performing comparison of catalytic properties and oligomeric states of functional PON1 in different enzyme preparations. Relevance of these data to understand in vivo physiological PON1 functioning is mandatory.
ESTHER : Renault_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1346
PubMedSearch : Renault_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1346
PubMedID: 19945920

Title : Structural approach to the aging of phosphylated cholinesterases - Masson_2010_Chem.Biol.Interact_187_157
Author(s) : Masson P , Nachon F , Lockridge O
Ref : Chemico-Biological Interactions , 187 :157 , 2010
Abstract : Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their reactivatability. This process, termed "aging", results from dealkylation of the adduct and depends on the structure of the organophosphyl moiety. Aged ChEs are resistant to reactivation by oximes. Owing to the toxicological importance of OPs, the molecular mechanism of aging has been the subject of research for decades. It was not clear whether aging involves the same bond breakage regardless the type of OP or is a scission of P-O-C bonds (P-O or O-C) in phosphates/phosphonates, P-N-C bonds in phosphoramidates, and P-S-C bonds in phosphonothionates. It was assumed that the resulting negatively charged atom on phosphorus of the aged adduct prevented nucleophilic attack by oximates, but studies on negatively charged model molecules do not support this hypothesis. Decrease in conformational flexibility of aged enzymes may contribute to their non-reactivatability by preventing proper adjustment of reactivators in the active site gorge. MALDI-TOF mass spectrometry of phosphylated human butyrylcholinesterase (hBChE) in water and in (18)O-water provided evidence that aging results from O-C breakage, i.e. O-dealkylation. In contrast, the isomalathion-BChE conjugate ages mostly through P-S bond cleavage, but a minor product results from O-C and/or S-C breakage. The crystal structures of hBChE and hAChE inhibited by tabun showed that aging of tabun-ChE conjugates results from O-dealkylation. However, depending on the nature of O-alkyl and N-alkyl chains, aging of BChE inhibited by other phosphoramidates results either from O-C breakage or deamination, i.e. P-N breakage. It was found that dealkylation of branched alkoxy involves a transient carbocation. Dealkylation of OP-ChE conjugates is accompanied by enzyme conformational changes. Urea, organic solvent, heat and pressure denaturation of human BChE showed that the conformational stability of aged OP-BChE conjugates is dramatically increased compared to native enzyme. Determination of the three-dimensional structure of BChE and AChE conjugated to different OPs showed that aged adducts form a salt bridge with the protonated catalytic histidine. Structure alteration of aged enzymes is accompanied by exit of water molecules from the enzyme's active site gorge. In addition, neutron scattering studies provided evidence that the structural dynamics of aged BChE is dramatically altered compared to native enzyme. Knowledge of the molecular basis of aging will help to design reactivators of aged ChEs, molecules capable of slowing the aging process, and pseudocatalytic ChE-based bioscavengers.
ESTHER : Masson_2010_Chem.Biol.Interact_187_157
PubMedSearch : Masson_2010_Chem.Biol.Interact_187_157
PubMedID: 20338153

Title : Application of laccase-mediator system (LMS) for the degradation of organophosphorus compounds - Trovaslet-Leroy_2010_Chem.Biol.Interact_187_393
Author(s) : Trovaslet-Leroy M , Jolivalt C , Froment MT , Brasme B , Lefebvre B , Daveloose D , Nachon F , Masson P
Ref : Chemico-Biological Interactions , 187 :393 , 2010
Abstract : Degradation of organophosphorus compounds was achieved in the presence of purified fungal laccase from Trametes versicolor and a small molecular weight redox mediator (ABTS). This laccase-mediator system (LMS) catalyzed degradation of VX, PhX and VR while had no apparent effect on CVX, ecothiophate or demeton. Inhibition of ABTS oxidation was shown with VX, PhX, VR and CVX. Results with CVX suggest either no degradation subsequent to interaction with the laccase active site or the formation of a new toxic compound. PhX degradation was also monitored by mass spectroscopy, a method that allowed us to identify certain intermediates formed during OP degradation. Altogether, results underline the importance of the OP nitrogen atom at beta-position and of its substituents, even though the intimate mechanism of laccase-catalyzed degradation is not yet known.
ESTHER : Trovaslet-Leroy_2010_Chem.Biol.Interact_187_393
PubMedSearch : Trovaslet-Leroy_2010_Chem.Biol.Interact_187_393
PubMedID: 20149786

Title : Preparation and characterization of methoxy polyethylene glycol-conjugated phosphotriesterase as a potential catalytic bioscavenger against organophosphate poisoning - Jun_2010_Chem.Biol.Interact_187_380
Author(s) : Jun D , Musilova L , Link M , Loiodice M , Nachon F , Rochu D , Renault F , Masson P
Ref : Chemico-Biological Interactions , 187 :380 , 2010
Abstract : Bioscavengers are considered as promising antidotes against organophosphate poisoning. We focused on a bacterial phosphotriesterase (PTE) expressed in Escherichia coli. The main disadvantage of this non-human catalytic bioscavenger is its relatively short half-life in the organism and strong immunogenicity after repeated administration. Therefore, we prepared different methoxy polyethylene glycol (MPEG)-conjugated recombinant PTE as a potential catalytic bioscavenger with the aim to improve its biological properties. Enzyme was modified with two linear monofunctional MPEG derivatives with reactive aldehyde group of molecular weight 2 kDa and 5 kDa. We optimized reaction conditions (reagent ratios, temperature and duration of modification reaction) and we prepared homogeneous population of fully modified recombinant PTE with molecular weight around 52 kDa and 76 kDa, respectively. Modified PTE was characterized using SDS-PAGE and MALDI-TOF and by determining K(m) and V(max). We also investigated thermal stability of modified enzyme at 37 degrees C. Based on our results, for future in vivo evaluation of pharmacokinetics and pharmacodynamics properties, we selected recombinant PTE modified with 5 kDa MPEG aldehyde for its superior thermal stability.
ESTHER : Jun_2010_Chem.Biol.Interact_187_380
PubMedSearch : Jun_2010_Chem.Biol.Interact_187_380
PubMedID: 20230809

Title : Structural evidence that human acetylcholinesterase inhibited by tabun ages through O-dealkylation - Carletti_2010_J.Med.Chem_53_4002
Author(s) : Carletti E , Colletier JP , Dupeux F , Trovaslet M , Masson P , Nachon F
Ref : Journal of Medicinal Chemistry , 53 :4002 , 2010
Abstract : Tabun is a warfare agent that inhibits human acetylcholinesterase (hAChE) by rapid phosphylation of the catalytic serine. A time-dependent reaction occurs on the tabun adduct, leading to an "aged" enzyme, resistant to oxime reactivators. The aging reaction may proceed via either dealkylation or deamidation, depending on the stereochemistry of the phosphoramidyl adduct. We solved the X-ray structure of aged tabun-hAChE complexed with fasciculin II, and we show that aging proceeds through O-dealkylation, in agreement with the aging mechanism that we determined for tabun-inhibited human butyrylcholinesterase and mouse acetylcholinesterase. Noteworthy, aging and binding of fasciculin II lead to an improved thermostability, resulting from additional stabilizing interactions between the two subdomains that face each other across the active site gorge. This first structure of hAChE inhibited by a nerve agent provides structural insight into the inhibition and aging mechanisms and a structural template for the design of molecules capable of reactivating aged hAChE.
ESTHER : Carletti_2010_J.Med.Chem_53_4002
PubMedSearch : Carletti_2010_J.Med.Chem_53_4002
PubMedID: 20408548
Gene_locus related to this paper: human-ACHE

Title : Butyrylcholinesterase for protection from organophosphorus poisons: catalytic complexities and hysteretic behavior - Masson_2010_Arch.Biochem.Biophys_494_107
Author(s) : Masson P , Lockridge O
Ref : Archives of Biochemistry & Biophysics , 494 :107 , 2010
Abstract : Butyrylcholinesterase is a promiscuous enzyme that displays complex kinetic behavior. It is toxicologically important because it detoxifies organophosphorus poisons (OP) by making a covalent bond with the OP. The OP and the butyrylcholinesterase are both inactivated in the process. Inactivation of butyrylcholinesterase has no adverse effects. However, inactivation of acetylcholinesterase in nerve synapses can be lethal. OP-inhibited butyrylcholinesterase and acetylcholinesterase can be reactivated with oximes provided the OP has not aged. Strategies for preventing the toxicity of OP include (a) treatment with an OP scavenger, (b) reaction of non-aged enzyme with oximes, (c) reactivation of aged enzyme, (d) slowing down aging with peripheral site ligands, and (e) design of mutants that rapidly hydrolyze OP. Option (a) has progressed through phase I clinical trials with human butyrylcholinesterase. Option (b) is in routine clinical use. The others are at the basic research level. Butyrylcholinesterase displays complex kinetic behavior including activation by positively charged esters, ability to hydrolyze amides, and a lag time (hysteresis) preceding hydrolysis of benzoylcholine and N-methylindoxyl acetate. Mass spectrometry has identified new OP binding motifs on tyrosine and lysine in proteins that have no active site serine. It is proposed, but not yet proven, that low dose exposure involves OP modification of proteins that have no active site serine.
ESTHER : Masson_2010_Arch.Biochem.Biophys_494_107
PubMedSearch : Masson_2010_Arch.Biochem.Biophys_494_107
PubMedID: 20004171

Title : Tyrosines of human and mouse transferrin covalently labeled by organophosphorus agents: a new motif for binding to proteins that have no active site serine - Li_2009_Toxicol.Sci_107_144
Author(s) : Li B , Schopfer LM , Grigoryan H , Thompson CM , Hinrichs SH , Masson P , Lockridge O
Ref : Toxicol Sci , 107 :144 , 2009
Abstract : The expectation from the literature is that organophosphorus (OP) agents bind to proteins that have an active site serine. However, transferrin, a protein with no active site serine, was covalently modified in vitro by 0.5mM 10-fluoroethoxyphosphinyl-N-biotinamido pentyldecanamide, chlorpyrifos oxon, diisopropylfluorophosphate, dichlorvos, sarin, and soman. The site of covalent attachment was identified by analyzing tryptic peptides in the mass spectrometer. Tyr 238 and Tyr 574 in human transferrin and Tyr 238, Tyr 319, Tyr 429, Tyr 491, and Tyr 518 in mouse transferrin were labeled by OP. Tyrosine in the small synthetic peptide ArgTyrThrArg made a covalent bond with diisopropylfluorophosphate, chlorpyrifos oxon, and dichlorvos at pH 8.3. These results, together with our previous demonstration that albumin and tubulin bind OP on tyrosine, lead to the conclusion that OP bind covalently to tyrosine, and that OP binding to tyrosine is a new OP-binding residue. The OP-reactive tyrosines are activated by interaction with Arg or Lys. It is suggested that many proteins in addition to those already identified may be modified by OP on tyrosine. The extent to which tyrosine modification by OP can occur in vivo and the toxicological implications of such modifications require further investigation.
ESTHER : Li_2009_Toxicol.Sci_107_144
PubMedSearch : Li_2009_Toxicol.Sci_107_144
PubMedID: 18930948

Title : Crystallographic snapshots of nonaged and aged conjugates of soman with acetylcholinesterase, and of a ternary complex of the aged conjugate with pralidoxime - Sanson_2009_J.Med.Chem_52_7593
Author(s) : Sanson B , Nachon F , Colletier JP , Froment MT , Toker L , Greenblatt HM , Sussman JL , Ashani Y , Masson P , Silman I , Weik M
Ref : Journal of Medicinal Chemistry , 52 :7593 , 2009
Abstract : Organophosphate compounds (OP) are potent inhibitors of acetylcholinesterases (AChEs) and can cause lethal poisoning in humans. Inhibition of AChEs by the OP soman involves phosphonylation of the catalytic serine, and subsequent dealkylation produces a form known as the "aged" enzyme. The nonaged form can be reactivated to a certain extent by nucleophiles, such as pralidoxime (2-PAM), whereas aged forms of OP-inhibited AChEs are totally resistant to reactivation. Here, we solved the X-ray crystal structures of AChE from Torpedo californica (TcAChE) conjugated with soman before and after aging. The absolute configuration of the soman stereoisomer adduct in the nonaged conjugate is P(S)C(R). A structural reorientation of the catalytic His440 side chain was observed during the aging process. Furthermore, the crystal structure of the ternary complex of the aged conjugate with 2-PAM revealed that the orientation of the oxime function does not permit nucleophilic attack on the phosphorus atom, thus providing a plausible explanation for its failure to reactivate the aged soman/AChE conjugate. Together, these three crystal structures provide an experimental basis for the design of new reactivators.
ESTHER : Sanson_2009_J.Med.Chem_52_7593
PubMedSearch : Sanson_2009_J.Med.Chem_52_7593
PubMedID: 19642642
Gene_locus related to this paper: torca-ACHE

Title : Direct correlation between molecular dynamics and enzymatic stability: a comparative neutron scattering study of native human butyrylcholinesterase and its aged soman conjugate - Gabel_2009_Biophys.J_96_1489
Author(s) : Gabel F , Masson P , Froment MT , Doctor BP , Saxena A , Silman I , Zaccai G , Weik M
Ref : Biophysical Journal , 96 :1489 , 2009
Abstract : An incoherent elastic neutron scattering study of the molecular dynamics of native human butyrylcholinesterase and its "aged" soman-inhibited conjugate revealed a significant change in molecular flexibility on an angstrom-nanosecond scale as a function of temperature. The results were related to the stability of each state as established previously by differential scanning calorimetry. A striking relationship was found between the denaturation behavior and the molecular flexibility of the native and inhibited enzymes as a function of temperature. This was reflected in a quantitative correlation between the atomic mean-square displacements on an angstrom-nanosecond scale determined by neutron spectroscopy and the calorimetric specific heat. By the application of a simple two-state model that describes the transition from a folded to a denatured state, the denaturation temperatures of the native and the inhibited enzyme were correctly extracted from the atomic mean-square displacements. Furthermore, the transition entropy and enthalpy extracted from the model fit of the neutron data were, within the experimental accuracy, compatible with the values determined by differential scanning calorimetry.
ESTHER : Gabel_2009_Biophys.J_96_1489
PubMedSearch : Gabel_2009_Biophys.J_96_1489
PubMedID: 19217865

Title : Structure, activities and biomedical applications of human butyrylcholinesterase - Masson_2009_Protein.Pept.Lett_16_1215
Author(s) : Masson P , Carletti E , Nachon F
Ref : Protein Pept Lett , 16 :1215 , 2009
Abstract : Human butyrylcholinesterase (BuChE) is a serine enzyme present in most organs and plasma. No clear physiological function has yet been assigned to BuChE, but it is a pharmacologically and toxicologically important enzyme that plays a role in degradation of numerous ester-containing drugs and poisonous esters. Thus, BuChE-based bioscavengers are an alternative for prophylaxis and treatments of intoxications by these compounds. Also, BuChE has been integrated in biosensors for detection of organophosphorus compounds and other cholinesterase inhibitors.
ESTHER : Masson_2009_Protein.Pept.Lett_16_1215
PubMedSearch : Masson_2009_Protein.Pept.Lett_16_1215
PubMedID: 19508180

Title : Carbofuran poisoning detected by mass spectrometry of butyrylcholinesterase adduct in human serum - Li_2009_J.Appl.Toxicol_29_149
Author(s) : Li H , Ricordel I , Tong L , Schopfer LM , Baud F , Megarbane B , Maury E , Masson P , Lockridge O
Ref : J Appl Toxicol , 29 :149 , 2009
Abstract : Carbofuran is a pesticide whose acute toxicity is due to inhibition of acetylcholinesterase. Butyrylcholinesterase (BChE) in plasma is inhibited by carbofuran and serves as a biomarker of poisoning by carbofuran. The goal was to develop a method to positively identify poisoning by carbofuran. Sera from an attempted murder and an attempted suicide were analyzed for the presence of carbofuran adducts on BChE. The BChE from 1 ml of serum was rapidly purified on a 0.2 ml procainamide-Sepharose column. Speed was essential because the carbofuran-BChE adduct decarbamylates with a half-life of about 2 h. The partially purified BChE was boiled to denature the protein, thus stopping decarbamylation and making the protein vulnerable to digestion with trypsin. The labeled peptide was partially purified by HPLC before analysis by LC/MS/MS in the multiple reaction monitoring mode on the QTRAP 2000 mass spectrometer. Carbofuran was found to be covalently bound to Ser 198 of human BChE in serum samples from two poisoning cases. Multiple reaction monitoring triggered MS/MS spectra positively identified the carbofuran-BChE adduct. In conclusion a mass spectrometry method to identify carbofuran poisoning in humans has been developed. The method uses 1 ml of serum and detects low-level exposure associated with as little as 20% inhibition of plasma butyrylcholinesterase.
ESTHER : Li_2009_J.Appl.Toxicol_29_149
PubMedSearch : Li_2009_J.Appl.Toxicol_29_149
PubMedID: 18937214

Title : Structure-activity analysis of aging and reactivation of human butyrylcholinesterase inhibited by analogues of tabun. - Carletti_2009_Biochem.J_421_97
Author(s) : Carletti E , Aurbek N , Gillon E , Loiodice M , Nicolet Y , Fontecilla-Camps JC , Masson P , Thiermann H , Nachon F , Worek F
Ref : Biochemical Journal , 421 :97 , 2009
Abstract : hBChE [human BChE (butyrylcholinesterase)] naturally scavenges OPs (organophosphates). This bioscavenger is currently in Clinical Phase I for pretreatment of OP intoxication. Phosphylated ChEs (cholinesterases) can undergo a spontaneous time-dependent process called 'aging' during which the conjugate is dealkylated, leading to creation of an enzyme that cannot be reactivated. hBChE inhibited by phosphoramidates such as tabun displays a peculiar resistance to oxime-mediated reactivation. We investigated the basis of oxime resistance of phosphoramidyl-BChE conjugates by determining the kinetics of inhibition, reactivation (obidoxime {1,1'-(oxybis-methylene) bis[4-(hydroxyimino) methyl] pyridinium dichloride}, TMB-4(Trimedoxime) [1,3-trimethylene-bis(4-hydroxyiminomethylpyridinium) dibromide], HL 7 {1-[[[4-(aminocarbonyl) pyridinio]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl] pyridinium dimethanesulfonate)}, HI-6 {1-[[[4-(aminocarbonyl) pyridinio] methoxy] methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate} and aging, and the crystal structures of hBChE inhibited by different N-monoalkyl and N,N-dialkyl tabun analogues. The refined structures of aged hBChE conjugates show that aging proceeds through O-dealkylation of the P(R) enantiomer of N,N-diethyl and N-propyl analogues, with subsequent formation of a salt bridge preventing reactivation, similarly to a previous observation made on tabun-ChE conjugates. Interestingly, the N-methyl analogue projects its amino group towards the choline-binding pocket, so that aging proceeds through deamination. This orientation results from a preference of hBChE's acyl-binding pocket for larger than 2-atoms linear substituents. The correlation between the inhibitory potency and the N-monoalkyl chain length is related to increasingly optimized interactions with the acyl-binding pocket as shown by the X-ray structures. These kinetics and X-ray data lead to a structure-activity relationship that highlights steric and electronic effects of the amino substituent of phosphoramidate. This study provides the structural basis to design new oximes capable of reactivating phosphoramidyl-hBChE conjugates after intoxication, notably when hBChE is used as pretreatment, or to design BChE-based catalytic bioscavengers.
ESTHER : Carletti_2009_Biochem.J_421_97
PubMedSearch : Carletti_2009_Biochem.J_421_97
PubMedID: 19368529
Gene_locus related to this paper: human-BCHE

Title : A collaborative endeavor to design cholinesterase-based catalytic scavengers against toxic organophosphorus esters - Masson_2008_Chem.Biol.Interact_175_273
Author(s) : Masson P , Nachon F , Broomfield CA , Lenz DE , Verdier L , Schopfer LM , Lockridge O
Ref : Chemico-Biological Interactions , 175 :273 , 2008
Abstract : Wild-type human butyrylcholinesterase (BuChE) has proven to be an efficient bioscavenger for protection against nerve agent toxicity. Human acetylcholinesterase (AChE) has a similar potential. A limitation to their usefulness is that both cholinesterases (ChEs) react stoichiometrically with organophosphosphorus (OP) esters. Because OPs can be regarded as pseudo-substrates for which the dephosphylation rate constant is almost zero, several strategies have been attempted to promote the dephosphylation reaction. Oxime-mediated reactivation of phosphylated ChEs generates a turnover, but it is too slow to make pseudo-catalytic scavengers of pharmacological interest. Alternatively, it was hypothesized that ChEs could be converted into OP hydrolases by using rational site-directed mutagenesis based upon the crystal structure of ChEs. The idea was to introduce a nucleophile into the oxyanion hole, at an appropriate position to promote hydrolysis of the phospho-serine bond via a base catalysis mechanism. Such mutants, if they showed the desired catalytic and pharmacokinetic properties, could be used as catalytic scavengers. The first mutant of human BuChE that was capable of hydrolyzing OPs was G117H. It had a slow rate. Crystallographic study of the G117H mutant showed that hydrolysis likely occurs by activation of a water molecule rather than direct nucleophilic attack by H117. Numerous BuChE mutants were made later, but none of them was better than the G117H mutant at hydrolyzing OPs, with the exception of soman. Soman aged too rapidly to be hydrolyzed by G117H. Hydrolysis was however accomplished with the double mutant G117H/E197Q, which did not age after phosphonylation with soman. Multiple mutations in the active center of human and Bungarus AChE led to enzymes displaying low catalytic activity towards OPs and unwanted kinetic complexities. A new generation of human AChE mutants has been designed with the assistance of molecular modelling and computational methods. According to the putative water-activation mechanism of G117H BChE, a new histidine/aspartate dyad was introduced into the active center of human AChE at the optimum location for hydrolysis of the OP adduct. Additional mutations were made for optimizing activity of the new dyad. It is anticipated that these new mutants will have OP hydrolase activity.
ESTHER : Masson_2008_Chem.Biol.Interact_175_273
PubMedSearch : Masson_2008_Chem.Biol.Interact_175_273
PubMedID: 18508040

Title : Binding and hydrolysis of soman by human serum albumin - Li_2008_Chem.Res.Toxicol_21_421
Author(s) : Li B , Nachon F , Froment MT , Verdier L , Debouzy JC , Brasme B , Gillon E , Schopfer LM , Lockridge O , Masson P
Ref : Chemical Research in Toxicology , 21 :421 , 2008
Abstract : Human plasma and fatty acid free human albumin were incubated with soman at pH 8.0 and 25 degrees C. Four methods were used to monitor the reaction of albumin with soman: progressive inhibition of the aryl acylamidase activity of albumin, the release of fluoride ion from soman, 31P NMR, and mass spectrometry. Inhibition (phosphonylation) was slow with a bimolecular rate constant of 15 +/- 3 M(-1) min (-1). MALDI-TOF and tandem mass spectrometry of the soman-albumin adduct showed that albumin was phosphonylated on tyrosine 411. No secondary dealkylation of the adduct (aging) occurred. Covalent docking simulations and 31P NMR experiments showed that albumin has no enantiomeric preference for the four stereoisomers of soman. Spontaneous reactivation at pH 8.0 and 25 degrees C, measured as regaining of aryl acylamidase activity and decrease of covalent adduct (pinacolyl methylphosphonylated albumin) by NMR, occurred at a rate of 0.0044 h (-1), indicating that the adduct is quite stable ( t1/2 = 6.5 days). At pH 7.4 and 22 degrees C, the covalent soman-albumin adduct, measured by MALDI-TOF mass spectrometry, was more stable ( t1/2 = 20 days). Though the concentration of albumin in plasma is very high (about 0.6 mM), its reactivity with soman (phosphonylation and phosphotriesterase activity) is too slow to play a major role in detoxification of the highly toxic organophosphorus compound soman. Increasing the bimolecular rate constant of albumin for organophosphates is a protein engineering challenge that could lead to a new class of bioscavengers to be used against poisoning by nerve agents. Soman-albumin adducts detected by mass spectrometry could be useful for the diagnosis of soman exposure.
ESTHER : Li_2008_Chem.Res.Toxicol_21_421
PubMedSearch : Li_2008_Chem.Res.Toxicol_21_421
PubMedID: 18163544

Title : An unexpected plasma cholinesterase activity rebound after challenge with a high dose of the nerve agent VX - Dorandeu_2008_Toxicology_248_151
Author(s) : Dorandeu F , Foquin A , Briot R , Delacour C , Denis J , Alonso A , Froment MT , Renault F , Lallement G , Masson P
Ref : Toxicology , 248 :151 , 2008
Abstract : Organophosphorus chemical warfare agents (nerve agents) are to be feared in military operations as well as in terrorist attacks. Among them, VX (O-ethyl-S-[2-(diisopropylamino)ethyl] methylphosphonothioate) is a low volatility liquid that represents a percutaneous as well as an inhalation hazard if aerosolized. It is a potent irreversible cholinesterase (ChE) inhibitor that causes severe signs and symptoms, including respiratory dysfunction that stems from different mechanisms. VX-induced pulmonary oedema was previously reported in dogs but mechanisms involved are not well understood, and its clinical significance remains to be assessed. An experimental model was thus developed to study VX-induced cardiovascular changes and pulmonary oedema in isoflurane-anaesthetized swine. In the course of this study, we observed a fast and unexpected rebound of plasma ChE activity following inhibition provoked by the intravenous injection of 6 and 12 microg kg(-1) of VX. In whole blood ChE activity, the rebound could stay unnoticed. Further investigations showed that the rebound of plasma esterase activity was neither related to spontaneous reactivation of ChE nor to VX-induced increase in paraoxonase/carboxylesterase activities. A bias in Ellman assay, haemoconcentration or severe liver cytolysis were also ruled out. All in all, these results suggest that the rebound was likely due to the release of butyrylcholinesterase into the blood stream from ChE producing organs. Nature of the organ(s) and mechanisms involved in enzyme release will need further investigations as it may represent a mechanism of defence, i.e. VX scavenging, that could advantageously be exploited.
ESTHER : Dorandeu_2008_Toxicology_248_151
PubMedSearch : Dorandeu_2008_Toxicology_248_151
PubMedID: 18450356

Title : Pseudo-esterase activity of human albumin: slow turnover on tyrosine 411 and stable acetylation of 82 residues including 59 lysines - Lockridge_2008_J.Biol.Chem_283_22582
Author(s) : Lockridge O , Xue W , Gaydess A , Grigoryan H , Ding SJ , Schopfer LM , Hinrichs SH , Masson P
Ref : Journal of Biological Chemistry , 283 :22582 , 2008
Abstract : Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5-6 h there was partial acetylation of 16-17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with beta-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 degrees C was 61 +/- 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover.
ESTHER : Lockridge_2008_J.Biol.Chem_283_22582
PubMedSearch : Lockridge_2008_J.Biol.Chem_283_22582
PubMedID: 18577514

Title : Five tyrosines and two serines in human albumin are labeled by the organophosphorus agent FP-biotin - Ding_2008_Chem.Res.Toxicol_21_1787
Author(s) : Ding SJ , Carr J , Carlson JE , Tong L , Xue W , Li Y , Schopfer LM , Li B , Nachon F , Asojo OA , Thompson CM , Hinrichs SH , Masson P , Lockridge O
Ref : Chemical Research in Toxicology , 21 :1787 , 2008
Abstract : Tyrosine 411 of human albumin is an established site for covalent attachment of 10-fluoroethoxyphosphinyl- N-biotinamidopentyldecanamide (FP-biotin), diisopropylfluorophosphate, chlorpyrifos oxon, soman, sarin, and dichlorvos. This work investigated the hypothesis that other residues in albumin could be modified by organophosphorus agents (OP). Human plasma was aggressively treated with FP-biotin; plasma proteins were separated into high and low abundant portions using a proteome partitioning antibody kit, and the proteins were digested with trypsin. The FP-biotinylated tryptic peptides were isolated by binding to monomeric avidin beads. The major sites of covalent attachment identified by mass spectrometry were Y138, Y148, Y401, Y411, Y452, S232, and S287 of human albumin. Prolonged treatment of pure human albumin with chlorpyrifos oxon labeled Y138, Y150, Y161, Y401, Y411, and Y452. To identify the most reactive residue, albumin was treated for 2 h with DFP, FP-biotin, chlorpyrifos oxon, or soman, digested with trypsin or pepsin, and analyzed by mass spectrometry. The most reactive residue was always Tyr 411. Diethoxyphosphate-labeled Tyr 411 was stable for months at pH 7.4. These results will be useful in the development of specific antibodies to detect OP exposure and to engineer albumin for use as an OP scavenger.
ESTHER : Ding_2008_Chem.Res.Toxicol_21_1787
PubMedSearch : Ding_2008_Chem.Res.Toxicol_21_1787
PubMedID: 18707141

Title : Mass spectrometry identifies covalent binding of soman, sarin, chlorpyrifos oxon, diisopropyl fluorophosphate, and FP-biotin to tyrosines on tubulin: a potential mechanism of long term toxicity by organophosphorus agents - Grigoryan_2008_Chem.Biol.Interact_175_180
Author(s) : Grigoryan H , Schopfer LM , Thompson CM , Terry AV , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 175 :180 , 2008
Abstract : Chronic low dose exposure to organophosphorus poisons (OP) results in cognitive impairment. Studies in rats have shown that OP interfere with microtubule polymerization. Since microtubules are required for transport of nutrients from the nerve cell body to the nerve synapse, it has been suggested that disruption of microtubule function could explain the learning and memory deficits associated with OP exposure. Tubulin is a major constituent of microtubules. We tested the hypothesis that OP bind to tubulin by treating purified bovine tubulin with sarin, soman, chlorpyrifos oxon, diisopropylfluorophosphate, and 10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide (FP-biotin). Tryptic peptides were isolated and analyzed by mass spectrometry. It was found that OP bound to tyrosine 83 of alpha tubulin in peptide TGTYR, tyrosine 59 in beta tubulin peptide YVPR, tyrosine 281 in beta tubulin peptide GSQQYR, and tyrosine 159 in beta tubulin peptide EEYPDR. The OP reactive tyrosines are located either near the GTP binding site or within loops that interact laterally with protofilaments. It is concluded that OP bind covalently to tubulin, and that this binding could explain cognitive impairment associated with OP exposure.
ESTHER : Grigoryan_2008_Chem.Biol.Interact_175_180
PubMedSearch : Grigoryan_2008_Chem.Biol.Interact_175_180
PubMedID: 18502412

Title : Hysteresis of insect acetylcholinesterase - Badiou_2008_Chem.Biol.Interact_175_410
Author(s) : Badiou A , Froment MT , Fournier D , Masson P , Belzunces LP
Ref : Chemico-Biological Interactions , 175 :410 , 2008
Abstract : Pre-steady-state catalytic properties of insect acetylcholinesterase (AChE, EC 3.1.1.7) were studied with the neutral substrate N-methylindoxylacetate. Kinetics of soluble Apis mellifera and Drosophila melanogaster AChE forms showed lags (v(i)=0) before reaching the steady-state. Results were interpreted in terms of slow equilibrium between two conformational states E and E' of insect AChE. Hysteresis of insect AChE has been pointed out for the first time. The hysteretic behaviour was found to depend on the NMIA concentration and the nature of the enzyme. The maximum induction times (tau(max)) to reach the steady-state were 800 and 1000s with soluble AChE from A. mellifera and D.melanogaster, respectively. The orders of magnitude of the tau(max) were high and similar to human AChE and BuChE.
ESTHER : Badiou_2008_Chem.Biol.Interact_175_410
PubMedSearch : Badiou_2008_Chem.Biol.Interact_175_410
PubMedID: 18602377

Title : Kinetic analysis of effector modulation of butyrylcholinesterase-catalysed hydrolysis of acetanilides and homologous esters - Masson_2008_FEBS.J_275_2617
Author(s) : Masson P , Froment MT , Gillon E , Nachon F , Lockridge O , Schopfer LM
Ref : Febs J , 275 :2617 , 2008
Abstract : The effects of tyramine, serotonin and benzalkonium on the esterase and aryl acylamidase activities of wild-type human butyrylcholinesterase and its peripheral anionic site mutant, D70G, were investigated. The kinetic study was carried out under steady-state conditions with neutral and positively charged aryl acylamides [o-nitrophenylacetanilide, o-nitrotrifluorophenylacetanilide and m-(acetamido) N,N,N-trimethylanilinium] and homologous esters (o-nitrophenyl acetate and acetylthiocholine). Tyramine was an activator of hydrolysis for neutral substrates and an inhibitor of hydrolysis for positively charged substrates. The affinity of D70G for tyramine was lower than that of the wild-type enzyme. Tyramine activation of hydrolysis for neutral substrates by D70G was linear. Tyramine was found to be a pure competitive inhibitor of hydrolysis for positively charged substrates with both wild-type butyrylcholinesterase and D70G. Serotonin inhibited both esterase and aryl acylamidase activities for both positively charged and neutral substrates. Inhibition of wild-type butyrylcholinesterase was hyperbolic (i.e. partial) with neutral substrates and linear with positively charged substrates. Inhibition of D70G was linear with all substrates. A comparison of the effects of tyramine and serotonin on D70G versus the wild-type enzyme indicated that: (a) the peripheral anionic site is involved in the nonlinear activation and inhibition of the wild-type enzyme; and (b) in the presence of charged substrates, the ligand does not bind to the peripheral anionic site, so that ligand effects are linear, reflecting their sole interaction with the active site binding locus. Benzalkonium acted as an activator at low concentrations with neutral substrates. High concentrations of benzalkonium caused parabolic inhibition of the activity with neutral substrates for both wild-type butyrylcholinesterase and D70G, suggesting multiple binding sites. Benzalkonium caused linear, noncompetitive inhibition of the positively charged aryl acetanilide m-(acetamido) N,N,N-trimethylanilinium for D70G, and an unusual mixed-type inhibition/activation (alpha > beta > 1) for wild-type butyrylcholinesterase with this substrate. No fundamental difference was observed between the effects of ligands on the butyrylcholinesterase-catalysed hydrolysis of esters and amides. Thus, butyrylcholinesterase uses the same machinery, i.e. the catalytic triad S198/H448/E325, for the hydrolysis of both types of substrate. The differences in response to ligand binding depend on whether the substrates are neutral or positively charged, i.e. the differences depend on the function of the peripheral site in wild-type butyrylcholinesterase, or the absence of its function in the D70G mutant. The complex inhibition/activation effects of effectors, depending on the integrity of the peripheral anionic site, reflect the allosteric 'cross-talk' between the peripheral anionic site and the catalytic centre.
ESTHER : Masson_2008_FEBS.J_275_2617
PubMedSearch : Masson_2008_FEBS.J_275_2617
PubMedID: 18422653

Title : Fast affinity purification coupled with mass spectrometry for identifying organophosphate labeled plasma butyrylcholinesterase - Li_2008_Chem.Biol.Interact_175_68
Author(s) : Li H , Tong L , Schopfer LM , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 175 :68 , 2008
Abstract : Classical plasma butyrylcholinesterase (BChE) purification involves dialysis and multiple steps of chromatography. We describe a procainamide affinity gel purification scheme that takes 15-30 min to purify BChE from 1 ml plasma. The method uses a microfuge spin column to build a 0.2 ml procainamide affinity column. The eluted BChE contains 3-4 microg of 500-fold purified BChE, free from 99% of contaminating plasma proteins. The BChE was further purified by gel electrophoresis. Tryptic peptides from the BChE containing gel electrophoresis band were prepared by in-gel digestion, separated by reverse phase liquid chromatography and identified by mass spectrometry. The 29 residue active site tryptic peptide labeled with the nerve agents soman or sarin was identified.
ESTHER : Li_2008_Chem.Biol.Interact_175_68
PubMedSearch : Li_2008_Chem.Biol.Interact_175_68
PubMedID: 18586231

Title : Lamellipodin proline rich peptides associated with native plasma butyrylcholinesterase tetramers - Li_2008_Biochem.J_411_425
Author(s) : Li H , Schopfer LM , Masson P , Lockridge O
Ref : Biochemical Journal , 411 :425 , 2008
Abstract : BChE (butyrylcholinesterase) protects the cholinergic nervous system from organophosphorus nerve agents by scavenging these toxins. Recombinant human BChE produced from transgenic goat to treat nerve agent intoxication is currently under development. The therapeutic potential of BChE relies on its ability to stay in the circulation for a prolonged period, which in turn depends on maintaining tetrameric quaternary configuration. Native human plasma BChE consists of 98% tetramers and has a half-life (t((1/2))) of 11-14 days. BChE in the neuromuscular junctions and the central nervous system is anchored to membranes through interactions with ColQ (AChE-associated collagen tail protein) and PRiMA (proline-rich membrane anchor) proteins containing proline-rich domains. BChE prepared in cell culture is primarily monomeric, unless expressed in the presence of proline-rich peptides. We hypothesized that a poly-proline peptide is an intrinsic component of soluble plasma BChE tetramers, just as it is for membrane-bound BChE. We found that a series of proline-rich peptides was released from denatured human and horse plasma BChE. Eight peptides, with masses from 2072 to 2878 Da, were purified by HPLC and sequenced by electrospray ionization tandem MS and Edman degradation. All peptides derived from the same proline-rich core sequence PSPPLPPPPPPPPPPPPPPPPPPPPLP (mass 2663 Da) but varied in length at their N- and C-termini. The source of these peptides was identified through database searching as RAPH1 [Ras-associated and PH domains (pleckstrin homology domains)-containing protein 1; lamellipodin, gi:82581557]. A proline-rich peptide of 17 amino acids derived from lamellipodin drove the assembly of human BChE secreted from CHO (Chinese-hamster ovary) cells into tetramers. We propose that the proline-rich peptides organize the 4 subunits of BChE into a 340 kDa tetramer, by interacting with the C-terminal BChE tetramerization domain.
ESTHER : Li_2008_Biochem.J_411_425
PubMedSearch : Li_2008_Biochem.J_411_425
PubMedID: 18076380

Title : Structural basis for natural lactonase and promiscuous phosphotriesterase activities - Elias_2008_J.Mol.Biol_379_1017
Author(s) : Elias M , Dupuy J , Merone L , Mandrich L , Porzio E , Moniot S , Rochu D , Lecomte C , Rossi M , Masson P , Manco G , Chabriere E
Ref : Journal of Molecular Biology , 379 :1017 , 2008
Abstract : Organophosphates are the largest class of known insecticides, several of which are potent nerve agents. Consequently, organophosphate-degrading enzymes are of great scientific interest as bioscavengers and biodecontaminants. Recently, a hyperthermophilic phosphotriesterase (known as SsoPox), from the Archaeon Sulfolobus solfataricus, has been isolated and found to possess a very high lactonase activity. Here, we report the three-dimensional structures of SsoPox in the apo form (2.6 A resolution) and in complex with a quorum-sensing lactone mimic at 2.0 A resolution. The structure also reveals an unexpected active site topology, and a unique hydrophobic channel that perfectly accommodates the lactone substrate. Structural and mutagenesis evidence allows us to propose a mechanism for lactone hydrolysis and to refine the catalytic mechanism established for phosphotriesterases. In addition, SsoPox structures permit the correlation of experimental lactonase and phosphotriesterase activities and this strongly suggests lactonase activity as the cognate function of SsoPox. This example demonstrates that promiscuous activities probably constitute a large and efficient reservoir for the creation of novel catalytic activities.
ESTHER : Elias_2008_J.Mol.Biol_379_1017
PubMedSearch : Elias_2008_J.Mol.Biol_379_1017
PubMedID: 18486146

Title : Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation - Carletti_2008_J.Am.Chem.Soc_130_16011
Author(s) : Carletti E , Li H , Li B , Ekstrom F , Nicolet Y , Loiodice M , Gillon E , Froment MT , Lockridge O , Schopfer LM , Masson P , Nachon F
Ref : Journal of the American Chemical Society , 130 :16011 , 2008
Abstract : Human butyrylcholinesterase (hBChE) hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides, and nerve agents. Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE) by phosphorylation of the catalytic serine. Phosphylated cholinesterase (ChE) can undergo a spontaneous, time-dependent process called "aging", during which the OP-ChE conjugate is dealkylated. This leads to irreversible inhibition of the enzyme. The inhibition of ChEs by tabun and the subsequent aging reaction are of particular interest, because tabun-ChE conjugates display an extraordinary resistance toward most current oxime reactivators. We investigated the structural basis of oxime resistance for phosphoramidated ChE conjugates by determining the crystal structures of the non-aged and aged forms of hBChE inhibited by tabun, and by updating the refinement of non-aged and aged tabun-inhibited mouse AChE (mAChE). Structures for non-aged and aged tabun-hBChE were refined to 2.3 and 2.1 A, respectively. The refined structures of aged ChE conjugates clearly show that the aging reaction proceeds through O-dealkylation of the P(R) enantiomer of tabun. After dealkylation, the negatively charged oxygen forms a strong salt bridge with protonated His438N epsilon2 that prevents reactivation. Mass spectrometric analysis of the aged tabun-inhibited hBChE showed that both the dimethylamine and ethoxy side chains were missing from the phosphorus. Loss of the ethoxy is consistent with the crystallography results. Loss of the dimethylamine is consistent with acid-catalyzed deamidation during the preparation of the aged adduct for mass spectrometry. The reported 3D data will help in the design of new oximes capable of reactivating tabun-ChE conjugates.
ESTHER : Carletti_2008_J.Am.Chem.Soc_130_16011
PubMedSearch : Carletti_2008_J.Am.Chem.Soc_130_16011
PubMedID: 18975951
Gene_locus related to this paper: human-BCHE , mouse-ACHE

Title : Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate - Masson_2007_J.Enzyme.Inhib.Med.Chem_22_463
Author(s) : Masson P , Froment MT , Darvesh S , Schopfer LM , Lockridge O
Ref : J Enzyme Inhib Med Chem , 22 :463 , 2007
Abstract : Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k(cat) = 0.13 +/- 0.02 min(-1) and Ks = 0.67 +/- 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k(cat) = k2). Though the aryl acylamidase activity of albumin is low (k(cat)/Ks = 195 M(-1)min(-1)), because of its high concentration in human plasma (0.6-1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.
ESTHER : Masson_2007_J.Enzyme.Inhib.Med.Chem_22_463
PubMedSearch : Masson_2007_J.Enzyme.Inhib.Med.Chem_22_463
PubMedID: 17847714

Title : Kinetic analysis of butyrylcholinesterase-catalyzed hydrolysis of acetanilides - Masson_2007_Biochim.Biophys.Acta_1774_1139
Author(s) : Masson P , Froment MT , Gillon E , Nachon F , Darvesh S , Schopfer LM
Ref : Biochimica & Biophysica Acta , 1774 :1139 , 2007
Abstract : The aryl-acylamidase (AAA) activity of butyrylcholinesterase (BuChE) has been known for a long time. However, the kinetic mechanism of aryl-acylamide hydrolysis by BuChE has not been investigated. Therefore, the catalytic properties of human BuChE and its peripheral site mutant (D70G) toward neutral and charged aryl-acylamides were determined. Three neutral (o-nitroacetanilide, m-nitroacetanilide, o-nitrophenyltrifluoroacetamide) and one positively charged (3-(acetamido) N,N,N-trimethylanilinium, ATMA) acetanilides were studied. Hydrolysis of ATMA by wild-type and D70G enzymes showed a long transient phase preceding the steady state. The induction phase was characterized by a hysteretic "burst". This reflects the existence of two enzyme states in slow equilibrium with different catalytic properties. Steady-state parameters for hydrolysis of the three acetanilides were compared to catalytic parameters for hydrolysis of esters giving the same acetyl intermediate. Wild-type BuChE showed substrate activation while D70G displayed a Michaelian behavior with ATMA as with positively charged esters. Owing to the low affinity of BuChE for amide substrates, the hydrolysis kinetics of neutral amides was first order. Acylation was the rate-determining step for hydrolysis of aryl-acetylamide substrates. Slow acylation of the enzyme, relative to that by esters may, in part, be due suboptimal fit of the aryl-acylamides in the active center of BuChE. The hypothesis that AAA and esterase active sites of BuChE are non-identical was tested with mutant BuChE. It was found that mutations on the catalytic serine, S198C and S198D, led to complete loss of both activities. The silent variant (FS117) had neither esterase nor AAA activity. Mutation in the peripheral site (D70G) had the same effect on esterase and AAA activities. Echothiophate inhibited both activities identically. It was concluded that the active sites for esterase and AAA activities are identical, i.e. S198. This excludes any other residue present in the gorge for being the catalytic nucleophile pole.
ESTHER : Masson_2007_Biochim.Biophys.Acta_1774_1139
PubMedSearch : Masson_2007_Biochim.Biophys.Acta_1774_1139
PubMedID: 17690023

Title : Use of a 'caged' analogue to study the traffic of choline within acetylcholinesterase by kinetic crystallography - Colletier_2007_Acta.Crystallogr.D.Biol.Crystallogr_63_1115
Author(s) : Colletier JP , Royant A , Specht A , Sanson B , Nachon F , Masson P , Zaccai G , Sussman JL , Goeldner M , Silman I , Bourgeois D , Weik M
Ref : Acta Crystallographica D Biol Crystallogr , 63 :1115 , 2007
Abstract : Acetylcholinesterase plays a crucial role in nerve-impulse transmission at cholinergic synapses. The apparent paradox that it displays high turnover despite its active site being buried raises cogent questions as to how the traffic of substrates and products to and from the active site can occur so rapidly in such circumstances. Here, a kinetic crystallography strategy aimed at structurally addressing the issue of product traffic in acetylcholinesterase is presented, in which UV-laser-induced cleavage of a photolabile precursor of the enzymatic product analogue arsenocholine, 'caged' arsenocholine, is performed in a temperature-controlled X-ray crystallography regime. The 'caged' arsenocholine was shown to bind at both the active and peripheral sites of acetylcholinesterase. UV irradiation of a complex with acetylcholinesterase during a brief temperature excursion from 100 K to room temperature is most likely to have resulted in a decrease in occupancy by the caged compound. Microspectrophotometric experiments showed that the caged compound had indeed been photocleaved. It is proposed that a fraction of the arsenocholine molecules released within the crystal had been expelled from both the active and the peripheral sites. Partial q-weighted difference refinement revealed a relative movement of the two domains in acetylcholinesterase after photolysis and the room-temperature excursion, resulting in an increase in the active-site gorge volume of 30% and 35% in monomers A and B of the asymmetric unit, respectively. Moreover, an alternative route to the active-site gorge of the enzyme appeared to open. This structural characterization of acetylcholinesterase 'at work' is consistent with the idea that choline exits from the enzyme after catalysis either via the gorge or via an alternative 'backdoor' trajectory.
ESTHER : Colletier_2007_Acta.Crystallogr.D.Biol.Crystallogr_63_1115
PubMedSearch : Colletier_2007_Acta.Crystallogr.D.Biol.Crystallogr_63_1115
PubMedID: 18007027
Gene_locus related to this paper: torca-ACHE

Title : Matrix-assisted laser desorption\/ionization time-of-flight mass spectrometry assay for organophosphorus toxicants bound to human albumin at Tyr411 - Li_2007_Anal.Biochem_361_263
Author(s) : Li B , Schopfer LM , Hinrichs SH , Masson P , Lockridge O
Ref : Analytical Biochemistry , 361 :263 , 2007
Abstract : Our goal was to determine whether chlorpyrifos oxon, dichlorvos, diisopropylfluorophosphate (DFP), and sarin covalently bind to human albumin. Human albumin or plasma was treated with organophosphorus (OP) agent at alkaline pH, digested with pepsin at pH 2.3, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Two singly charged peaks m/z 1718 and 1831, corresponding to the unlabeled peptide fragments containing the active site Tyr411 residue, were detected in all samples. The sequences of the two peptides were VRYTKKVPQVSTPTL and LVRYTKKVPQVSTPTL. The peptide-OP adducts of these peptides were also found. They had masses of 1854 and 1967 for chlorpyrifos oxon, 1825 and 1938 for dichlorvos, 1881 and 1994 for DFP, and 1838 and 1938 for sarin; these masses fit a mechanism whereby OP bound covalently to Tyr411. The binding of DFP to Tyr411 of human albumin was confirmed by electrospray tandem mass spectrometry and analysis of product ions. None of the OP-albumin adducts lost an alkoxy group, leading to the conclusion that aging did not occur. Our results show that OP pesticides and nerve agents bind covalently to human albumin at Tyr411. The presence of Tyr411 on an exposed surface of albumin suggests that an antibody response could be generated against OP-albumin adducts.
ESTHER : Li_2007_Anal.Biochem_361_263
PubMedSearch : Li_2007_Anal.Biochem_361_263
PubMedID: 17188226

Title : Hydrolysis of oxo- and thio-esters by human butyrylcholinesterase - Masson_2007_Biochim.Biophys.Acta_1774_16
Author(s) : Masson P , Froment MT , Gillon E , Nachon F , Lockridge O , Schopfer LM
Ref : Biochimica & Biophysica Acta , 1774 :16 , 2007
Abstract : Catalytic parameters of human butyrylcholinesterase (BuChE) for hydrolysis of homologous pairs of oxo-esters and thio-esters were compared. Substrates were positively charged (benzoylcholine versus benzoylthiocholine) and neutral (phenylacetate versus phenylthioacetate). In addition to wild-type BuChE, enzymes containing mutations were used. Single mutants at positions: G117, a key residue in the oxyanion hole, and D70, the main component of the peripheral anionic site were tested. Double mutants containing G117H and mutations on residues of the oxyanion hole (G115, A199), or the pi-cation binding site (W82), or residue E197 that is involved in stabilization of tetrahedral intermediates were also studied. A mathematical analysis was used to compare data for BuChE-catalyzed hydrolysis of various pairs of oxo-esters and thio-esters and to determine the rate-limiting step of catalysis for each substrate. The interest and limitation of this method is discussed. Molecular docking was used to analyze how the mutations could have altered the binding of the oxo-ester or the thio-ester. Results indicate that substitution of the ethereal oxygen for sulfur in substrates may alter the adjustment of substrate in the active site and stabilization of the transition-state for acylation. This affects the k2/k3 ratio and, in turn, controls the rate-limiting step of the hydrolytic reaction. Stabilization of the transition state is modulated both by the alcohol and acyl moieties of substrate. Interaction of these groups with the ethereal hetero-atom can have a neutral, an additive or an antagonistic effect on transition state stabilization, depending on their molecular structure, size and enantiomeric configuration.
ESTHER : Masson_2007_Biochim.Biophys.Acta_1774_16
PubMedSearch : Masson_2007_Biochim.Biophys.Acta_1774_16
PubMedID: 17182295

Title : Stabilization of the active form(s) of human paraoxonase by human phosphate-binding protein - Rochu_2007_Biochem.Soc.Trans_35_1616
Author(s) : Rochu D , Chabriere E , Renault F , Elias M , Clery-Barraud C , Masson P
Ref : Biochemical Society Transactions , 35 :1616 , 2007
Abstract : While there is a consensus that human PON1 (paraoxonase-1) has a protective role, its primary biological function remains unclear. A protective role against poisoning by organophosphates [OPs (organophosphorus compounds)] drove earlier works. Clinical interest has recently focused on a protective role of PON1 against vascular diseases. PON1 resides mainly on HDL (high-density lipoprotein) particles, and converging recent works show that both its activities and stability dramatically depend on this versatile and dynamic molecular environment. The discovery that HPBP (human phosphate-binding protein) has a firm tendency to associate with PON1 has steered new directions for characterizing PON1 functional state(s). Storage stability studies provided evidence that HPBP is involved in maintaining physiologically active PON1 conformation(s). Thermal stability studies showed that human PON1 is remarkably thermostable and that its association with HPBP strongly contributes to slowing down the denaturation rate. A hybrid PON1, displaying mutations that stabilized recombinant enzyme expressed in Escherichia coli, was shown to be more thermostable than natural human PON1. Predictably, its stability was unaffected by the presence of HPBP. Synergistic efforts on characterizing natural PON1 and rPON1 (recombinant PON1) provide information for the design of future stable mutants of PON1-based bioscavengers to be used as safe and effective countermeasures to challenge OPs. Maintaining a stable environment for such administrable human rPON1 should, at least, preserve the anti-atherogenic activity of the enzyme.
ESTHER : Rochu_2007_Biochem.Soc.Trans_35_1616
PubMedSearch : Rochu_2007_Biochem.Soc.Trans_35_1616
PubMedID: 18031277

Title : Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry - Li_2007_Toxicol.Sci_100_136
Author(s) : Li H , Schopfer LM , Nachon F , Froment MT , Masson P , Lockridge O
Ref : Toxicol Sci , 100 :136 , 2007
Abstract : Some organophosphorus compounds are toxic because they inhibit acetylcholinesterase (AChE) by phosphylation of the active site serine, forming a stable conjugate: Ser-O-P(O)-(Y)-(XR) (where X can be O, N, or S and Y can be methyl, OR, or SR). The inhibited enzyme can undergo an aging process, during which the X-R moiety is dealkylated by breaking either the P-X or the X-R bond depending on the specific compound, leading to a nonreactivatable enzyme. Aging mechanisms have been studied primarily using AChE. However, some recent studies have indicated that organophosphate-inhibited butyrylcholinesterase (BChE) may age through an alternative pathway. Our work utilized matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to study the aging mechanism of human BChE inhibited by dichlorvos, echothiophate, diisopropylfluorophosphate (DFP), isomalathion, soman, sarin, cyclohexyl sarin, VX, and VR. Inhibited BChE was aged in the presence of H2O18 to allow incorporation of (18)O, if cleavage was at the P-X bond. Tryptic-peptide organophosphate conjugates were identified through peptide mass mapping. Our results showed no aging of VX- and VR-treated BChE at 25 degrees C, pH 7.0. However, BChE inhibited by dichlorvos, echothiophate, DFP, soman, sarin, and cyclohexyl sarin aged exclusively through O-C bond cleavage, i.e., the classical X-R scission pathway. In contrast, isomalathion aged through both X-R and P-X pathways; the main aged product resulted from P-S bond cleavage and a minor product resulted from O-C and/or S-C bond cleavage.
ESTHER : Li_2007_Toxicol.Sci_100_136
PubMedSearch : Li_2007_Toxicol.Sci_100_136
PubMedID: 17698511

Title : Capillary electrophoresis versus differential scanning calorimetry for the analysis of free enzyme versus enzyme-ligand complexes: in the search of the ligand-free status of cholinesterases - Rochu_2006_Electrophoresis_27_442
Author(s) : Rochu D , Clery-Barraud C , Renault F , Chevalier A , Bon C , Masson P
Ref : Electrophoresis , 27 :442 , 2006
Abstract : Cholinesterases (ChEs) are highly efficient biocatalysts whose active site is buried in a deep, narrow gorge. The talent of CE to discover inhibitors in the gorge of highly purified preparations has fairly altered the meaning of a ChE ligand-free status. To attempt at a description of this one, we investigated the stability of Bungarus fasciatus acetylcholinesterase (AChE), alone or complexed with different inhibitors. Determination of mid-transition temperature for thermal denaturation, using differential scanning calorimetry (DSC) and CE, provided conflicting results. Discrepancies strongly question the reality of a ligand-free AChE state. DSC allowed estimation of the stability of AChE-ligands complexes, and to rank the stabilizing effect of different inhibitors. CE acted as a detector of hidden ligands, provided that they were charged, reversibly bound, and thus dissociable upon action of electric fields. Then, CE allowed quantification of the stability of ligand-free AChE. CE and DSC providing each fractional and nonredundant information, cautious attention must be paid for actual estimation of the conformational stability of ChEs. Because inhibitors used in purification of ChEs by affinity chromatography are charged, CE remains a leading method to estimate enzyme stability and detect the presence of bound hidden ligands.
ESTHER : Rochu_2006_Electrophoresis_27_442
PubMedSearch : Rochu_2006_Electrophoresis_27_442
PubMedID: 16342323

Title : Rat butyrylcholinesterase-catalysed hydrolysis of N-alkyl homologues of benzoylcholine - Hrabovska_2006_FEBS.J_273_1185
Author(s) : Hrabovska A , Debouzy JC , Froment MT , Devinsky F , Paulikova I , Masson P
Ref : Febs J , 273 :1185 , 2006
Abstract : The purpose of this work was to study the catalytic properties of rat butyrylcholinesterase with benzoylcholine (BzCh) and N-alkyl derivatives of BzCh (BCHn) as substrates. Complex hysteretic behaviour was observed in the approach to steady-state kinetics for each ester. Hysteresis consisted of a long lag phase with damped oscillation. The presence of a long lag phase, with no oscillations, in substrate hydrolysis by rat butyrylcholinesterase was also observed with N-methylindoxyl acetate as substrate. Hysteretic behaviour was explained by the existence of two interconvertible butyrylcholinesterase forms in slow equilibrium, while just one of them is catalytically active. The damped oscillations were explained by the existence of different substrate conformational states and/or aggregates (micelles) in slow equilibrium. Different substrate conformational states were confirmed by 1H-NMR. The K(m) values for substrates decreased as the length of the alkyl chain increased. High affinity of the enzyme for the longest alkyl chain length substrates was explained by multiple hydrophobic interactions of the alkyl chain with amino acid residues lining the active site gorge. Molecular modelling studies supported this interpretation; docking energy decreased as the length of the alkyl chain increased. The long-chain substrates had reduced k(cat) values. Docking studies showed that long-chain substrates were not optimally oriented in the active site for catalysis, thus explaining the slow rate of hydrolysis. The hydrolytic rate of BCH12 and longer alkyl chain esters vs. substrate concentration showed a premature plateau far below V(max). This was due to the loss of substrate availability. The best substrates for rat butyrylcholinesterase were short alkyl homologues, BzCh - BCH4.
ESTHER : Hrabovska_2006_FEBS.J_273_1185
PubMedSearch : Hrabovska_2006_FEBS.J_273_1185
PubMedID: 16519684

Title : Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters - Poyot_2006_Biochim.Biophys.Acta_1764_1470
Author(s) : Poyot T , Nachon F , Froment MT , Loiodice M , Wieseler S , Schopfer LM , Lockridge O , Masson P
Ref : Biochimica & Biophysica Acta , 1764 :1470 , 2006
Abstract : Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.
ESTHER : Poyot_2006_Biochim.Biophys.Acta_1764_1470
PubMedSearch : Poyot_2006_Biochim.Biophys.Acta_1764_1470
PubMedID: 16962835

Title : Improved access to 2-O-monobenzyl ethers of beta-cyclodextrin as precursors of catalysts for organophosphoryl esters hydrolysis - Masurier_2006_Carbohydr.Res_341_935
Author(s) : Masurier N , Estour F , Lefevre B , Brasme B , Masson P , Lafont O
Ref : Carbohydr Res , 341 :935 , 2006
Abstract : A comparative study of reaction conditions was performed for the synthesis of a 2-O-monobenzyl ether of cyclomaltoheptaose (beta-CD). Optimal conditions involved sodium ethoxide in Me(2)SO and benzyl bromide. The methodology was extended to the preparation of various 2(I)-O-iodobenzyl and 2(I)-O-carboxymethylbenzyl derivatives of beta-CD including a 3-carboxymethyl-4-iodobenzyl derivative of interest as precursor of an enzyme mimic to degrade the organophosphoryl ester diethyl 4-nitrophenyl phosphate (paraoxon).
ESTHER : Masurier_2006_Carbohydr.Res_341_935
PubMedSearch : Masurier_2006_Carbohydr.Res_341_935
PubMedID: 16530173

Title : Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging - Nachon_2005_Biochemistry_44_1154
Author(s) : Nachon F , Asojo OA , Borgstahl GE , Masson P , Lockridge O
Ref : Biochemistry , 44 :1154 , 2005
Abstract : Organophosphorus poisons (OP) bind covalently to the active-site serine of cholinesterases. The inhibited enzyme can usually be reactivated with powerful nucleophiles such as oximes. However, the covalently bound OP can undergo a suicide reaction (termed aging) yielding nonreactivatable enzyme. In human butyrylcholinesterase (hBChE), aging involves the residues His438 and Glu197 that are proximal to the active-site serine (Ser198). The mechanism of aging is known in detail for the nerve gases soman, sarin, and tabun as well as the pesticide metabolite isomalathion. Aging of soman- and sarin-inhibited acetylcholinesterase occurs by C-O bond cleavage, whereas that of tabun- and isomalathion-inhibited acetylcholinesterase occurs by P-N and P-S bond cleavage, respectively. In this work, the crystal structures of hBChE inhibited by the ophthalmic reagents echothiophate (nonaged and aged) and diisopropylfluorophosphate (aged) were solved and refined to 2.1, 2.25, and 2.2 A resolution, respectively. No appreciable shift in the position of the catalytic triad histidine was observed between the aged and nonaged conjugates of hBChE. This absence of shift contrasts with the aged and nonaged crystal structures of Torpedo californica acetylcholinesterase inhibited by the nerve agent VX. The nonaged hBChE structure shows one water molecule interacting with Glu197 and the catalytic triad histidine (His438). Interestingly, this water molecule is ideally positioned to promote aging by two mechanisms: breaking either a C-O bond or a P-O bond. Pesticides and certain stereoisomers of nerve agents are expected to undergo aging by breaking the P-O bond.
ESTHER : Nachon_2005_Biochemistry_44_1154
PubMedSearch : Nachon_2005_Biochemistry_44_1154
PubMedID: 15667209
Gene_locus related to this paper: human-BCHE

Title : The powerful high pressure tool for protein conformational studies - Marchal_2005_Braz.J.Med.Biol.Res_38_1175
Author(s) : Marchal S , Torrent J , Masson P , Kornblatt JM , Tortora P , Fusi P , Lange R , Balny C
Ref : Brazilian Journal of Medical & Biological Research , 38 :1175 , 2005
Abstract : The pressure behavior of proteins may be summarized as a the pressure-induced disordering of their structures. This thermodynamic parameter has effects on proteins that are similar but not identical to those induced by temperature, the other thermodynamic parameter. Of particular importance are the intermolecular interactions that follow partial protein unfolding and that give rise to the formation of fibrils. Because some proteins do not form fibrils under pressure, these observations can be related to the shape of the stability diagram. Weak interactions which are differently affected by hydrostatic pressure or temperature play a determinant role in protein stability. Pressure acts on the 2 degrees, 3 degrees and 4 degrees structures of proteins which are maintained by electrostatic and hydrophobic interactions and by hydrogen bonds. We present some typical examples of how pressure affects the tertiary structure of proteins (the case of prion proteins), induces unfolding (ataxin), is a convenient tool to study enzyme dissociation (enolase), and provides arguments to understand the role of the partial volume of an enzyme (butyrylcholinesterase). This approach may have important implications for the understanding of the basic mechanism of protein diseases and for the development of preventive and therapeutic measures.
ESTHER : Marchal_2005_Braz.J.Med.Biol.Res_38_1175
PubMedSearch : Marchal_2005_Braz.J.Med.Biol.Res_38_1175
PubMedID: 16082457

Title : Effects of soman inhibition and of structural differences on cholinesterase molecular dynamics: a neutron scattering study - Gabel_2005_Biophys.J_89_3303
Author(s) : Gabel F , Weik M , Masson P , Renault F , Fournier D , Brochier L , Doctor BP , Saxena A , Silman I , Zaccai G
Ref : Biophysical Journal , 89 :3303 , 2005
Abstract : Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.
ESTHER : Gabel_2005_Biophys.J_89_3303
PubMedSearch : Gabel_2005_Biophys.J_89_3303
PubMedID: 16100272

Title : [Butyrylcholinesterase: 3D structure, catalytic mechanisms] - Nachon_2005_Ann.Pharm.Fr_63_194
Author(s) : Nachon F , Nicolet Y , Masson P
Ref : Ann Pharm Fr , 63 :194 , 2005
Abstract : Cholinesterases are the main targets of organophosphorus compounds. Although progresses in prophylaxis and treatment of nerve agent poisoning have been achieved in the past twenty years, pharmacological protection and emergency treatment remain imperfect. All classical pharmacological ways have been explored and, the current medical counter-measure arsenal is generally not expected to change much. However, the emergence of the catalytic bioscavenger concept has aroused new hope. In addition, resolution of the three-dimensional structure of human butyrylcholinesterase (BChE) answered some fundamental issues in medical defense research: 1) a better description of cholinesterase catalytic mechanisms (substrate hydrolysis, inhibition by organophosphorus); 2) aging mechanism of phosphylated cholinesterases caused by the dealkylation of branched organophosphorous moiety; 3) reasoned site-directed mutagenesis of human BChE with the aim of making operational mutants capable of detoxifying organophosphorus substrates.
ESTHER : Nachon_2005_Ann.Pharm.Fr_63_194
PubMedSearch : Nachon_2005_Ann.Pharm.Fr_63_194
PubMedID: 15976688

Title : Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma - Li_2005_Biochem.Pharmacol_70_1673
Author(s) : Li B , Sedlacek M , Manoharan I , Boopathy R , Duysen EG , Masson P , Lockridge O
Ref : Biochemical Pharmacology , 70 :1673 , 2005
Abstract : The goal of this work was to identify the esterases in human plasma and to clarify common misconceptions. The method for identifying esterases was nondenaturing gradient gel electrophoresis stained for esterase activity. We report that human plasma contains four esterases: butyrylcholinesterase (EC 3.1.1.8), paraoxonase (EC 3.1.8.1), acetylcholinesterase (EC 3.1.1.7), and albumin. Butyrylcholinesterase (BChE), paraoxonase (PON1), and albumin are in high enough concentrations to contribute significantly to ester hydrolysis. However, only trace amounts of acetylcholinesterase (AChE) are present. Monomeric AChE is seen in wild-type as well as in silent BChE plasma. Albumin has esterase activity with alpha- and beta-naphthylacetate as well as with p-nitrophenyl acetate. Misconception #1 is that human plasma contains carboxylesterase. We demonstrate that human plasma contains no carboxylesterase (EC 3.1.1.1), in contrast to mouse, rat, rabbit, horse, cat, and tiger that have high amounts of plasma carboxylesterase. Misconception #2 is that lab animals have BChE but no AChE in their plasma. We demonstrate that mice, unlike humans, have substantial amounts of soluble AChE as well as BChE in their plasma. Plasma from AChE and BChE knockout mice allowed identification of AChE and BChE bands without the use of inhibitors. Human BChE is irreversibly inhibited by diisopropylfluorophosphate, echothiophate, and paraoxon, but mouse BChE spontaneously reactivates. Since human plasma contains no carboxylesterase, only BChE, PON1, and albumin esterases need to be considered when evaluating hydrolysis of an ester drug in human plasma.
ESTHER : Li_2005_Biochem.Pharmacol_70_1673
PubMedSearch : Li_2005_Biochem.Pharmacol_70_1673
PubMedID: 16213467

Title : Structural data on the aging of diethylphosphoryl-butyrylcholinesterase -
Author(s) : Nachon F , Asojo OA , Borgstahl GE , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 157-158 :408 , 2005
PubMedID: 16429549

Title : The reactant state for substrate-activated turnover of acetylthiocholine by butyrylcholinesterase is a tetrahedral intermediate - Tormos_2005_J.Am.Chem.Soc_127_14538
Author(s) : Tormos JR , Wiley KL , Seravalli J , Nachon F , Masson P , Nicolet Y , Quinn DM
Ref : Journal of the American Chemical Society , 127 :14538 , 2005
Abstract : Secondary beta-deuterium kinetic isotope effects have been measured as a function of substrate concentration for recombinant human butyrylcholinesterase-catalyzed hydrolysis of acetyl-L3-thiocholine (L = 1H or 2H). The isotope effect on V/K is inverse, D3V/K = 0.93 +/- 0.03, which is consistent with conversion of the sp2 hybridized carbonyl carbon of the scissile ester bond of the E + A reactant state to a quasi-tetrahedral structure in the acylation transition state. In contrast, the isotope effect on Vmax under conditions of substrate activation is markedly normal, D3(betaVmax) = 1.29 +/- 0.06, an observation that is consistent with accumulation of a tetrahedral intermediate as the reactant state for catalytic turnover. Generally, tetrahedral intermediates for nonenzymatic ester hydrolyses are high-energy steady-state intermediates. Apparently, butyrylcholinesterase displays an unusual ability to stabilize such intermediates. Hence, the catalytic power of cholinesterases can largely be understood in terms of their ability to stabilize tetrahedral intermediates in the multistep reaction mechanism.
ESTHER : Tormos_2005_J.Am.Chem.Soc_127_14538
PubMedSearch : Tormos_2005_J.Am.Chem.Soc_127_14538
PubMedID: 16231883

Title : Synthesis of 2-substituted beta-cyclodextrin derivatives with a hydrolytic activity against the organophosphorylester paraoxon - Masurier_2005_Eur.J.Med.Chem_40_615
Author(s) : Masurier N , Estour F , Froment MT , Lefevre B , Debouzy JC , Brasme B , Masson P , Lafont O
Ref : Eur Journal of Medicinal Chemistry , 40 :615 , 2005
Abstract : Beta-cyclodextrin was substituted by an iodosobenzoic acid derivative to create a catalytic hydrolytic activity against neurotoxic organophosphorus agents. The catalytic moiety was introduced on a secondary hydroxy group at the position 2 of a glucose unit. Several beta-cyclodextrin derivatives were obtained. In these derivatives, the methylene linker occupied all potential positions on the aromatic ring. Kinetic assays were carried out with paraoxon as organophosphate model. Three regioisomers hydrolyzed paraoxon, although the paraoxon-leaving group, para-nitrophenol, was not released from the beta-cyclodextrin torus.
ESTHER : Masurier_2005_Eur.J.Med.Chem_40_615
PubMedSearch : Masurier_2005_Eur.J.Med.Chem_40_615
PubMedID: 15935896

Title : Hysteresis of butyrylcholinesterase in the approach to steady-state kinetics - Masson_2005_Chem.Biol.Interact_157-158_143
Author(s) : Masson P , Schopfer LM , Froment MT , Debouzy JC , Nachon F , Gillon E , Lockridge O , Hrabovska A , Goldstein BN
Ref : Chemico-Biological Interactions , 157-158 :143 , 2005
Abstract : Butyrylcholinesterase (BChE) displays hysteretic behavior with certain neutral and charged substrates in the approach to steady state. Previous studies led us to interpret this phenomenon in terms of slow transitions between two enzyme conformers E and E'. This kinetic peculiarity is observed in human, horse and rat BChE. Oscillations that superimpose on the hysteretic lag are observed when benzoylcholine and N-alkyl derivatives of benzoylcholine are used as substrate. Hysteresis of BChE can be modulated by medium parameters (pH, salts, temperature, and pressure). Though mutant enzymes show different hysteretic behavior, so far attempts to provide a molecular mechanism of BChE hysteresis from mutagenesis studies have been unproductive. However, the substrate dependence of the hysteretic induction times, using wild-type BChE and several mutants, allowed us to build a general, mechanistic model for the hysteresis. In this model, substrate can bind to E, E', or both conformers, and ES and/or E'S can be catalytically active. The exact pathway followed depends on both the nature of the substrate and the structure of the BChE mutant under study. We propose that oscillations develop when substrate exists in different, slowly interconvertible, conformational and/or aggregation forms, of which only the minor form is capable of reacting with BChE. In support of this proposal, NMR studies have provided direct evidence for slow equilibria between monomeric and micellar forms of long-chain, alkyl derivatives of benzoyl-(N-substituted) choline. There is no direct evidence that hysteresis plays a role in BChE function(s). However, the "new view" of protein dynamics proposes that proteins are normally in equilibrium between pre-existing, functional and non-functional conformers; and that binding a ligand to the functional form shifts that equilibrium towards the functional conformation. Therefore, a physiological or toxicological relevance for the hysteresis in BChE cannot be ruled out.
ESTHER : Masson_2005_Chem.Biol.Interact_157-158_143
PubMedSearch : Masson_2005_Chem.Biol.Interact_157-158_143
PubMedID: 16256969

Title : Contribution of the active-site metal cation to the catalytic activity and to the conformational stability of phosphotriesterase: temperature- and pH-dependence - Rochu_2004_Biochem.J_380_627
Author(s) : Rochu D , Viguie N , Renault F , Crouzier D , Froment MT , Masson P
Ref : Biochemical Journal , 380 :627 , 2004
Abstract : Phosphotriesterase (PTE) detoxifies nerve agents and organophosphate pesticides. The two zinc cations of the PTE active centre can be substituted by other transition metal cations without loss of activity. Furthermore, metal-substituted PTEs display differences in catalytic properties. A prerequisite for engineering highly efficient mutants of PTE is to improve their thermostability. Isoelectric focusing, capillary electrophoresis and steady-state kinetics analysis were used to determine the contribution of the active-site cations Zn2+, Co2+ or Cd2+ to both the catalytic activity and the conformational stability of the corresponding PTE isoforms. The three isoforms have different pI values (7.2, 7.5 and 7.1) and showed non-superimposable electrophoretic titration curves. The overall structural alterations, causing changes in functional properties, were found to be related to the nature of the bound cation: ionic radius and ion electronegativity correlate with Km and kcat respectively. In addition, the pH-dependent activity profiles of isoforms were different. The temperature-dependent profiles of activity showed maximum activity at T < or =35 degrees C, followed by an activation phase near 45-48 degrees C and then inactivation which was completed at 60 degrees C. Analysis of thermal denaturation of the PTEs provided evidence that the activation phase resulted from a transient intermediate. Finally, at the optimum activity between pH 8 and 9.4, the thermostability of the different PTEs increased as the pH decreased, and the metal cation modulated stability (Zn2+-, Co2+- and Cd2+-PTE showed different T (m) values of 60.5-67 degrees C, 58-64 degrees C and 53-64 degrees C respectively). Requirements for optimum activity of PTE (displayed by Co2+-PTE) and maximum stability (displayed by Zn2+-PTE) were demonstrated.
ESTHER : Rochu_2004_Biochem.J_380_627
PubMedSearch : Rochu_2004_Biochem.J_380_627
PubMedID: 15018612

Title : Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants - Wang_2004_Anal.Biochem_329_131
Author(s) : Wang Y , Schopfer LM , Duysen EG , Nachon F , Masson P , Lockridge O
Ref : Analytical Biochemistry , 329 :131 , 2004
Abstract : Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1 mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5 microg/ml human G117H BChE, 2 microg/ml wild-type mouse BChE, and 0.06 microg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1 mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.
ESTHER : Wang_2004_Anal.Biochem_329_131
PubMedSearch : Wang_2004_Anal.Biochem_329_131
PubMedID: 15136175

Title : Poster (49) Crystallographic basis for substrate\/product exchange in cholinesterases. -
Author(s) : Nicolet Y , Lockridge O , Masson P , Fontecilla-Camps JC , Nachon F
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :347 , 2004
PubMedID:

Title : Rate-determining step of butyrylcholinesterase-catalyzed hydrolysis of benzoylcholine and benzoylthiocholine. Volumetric study of wild-type and D70G mutant behavior - Masson_2004_Eur.J.Biochem_271_1980
Author(s) : Masson P , Bec N , Froment MT , Nachon F , Balny C , Lockridge O , Schopfer LM
Ref : European Journal of Biochemistry , 271 :1980 , 2004
Abstract : The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BCHE) is deacylation (k(3)), whereas it is acylation (k(2)) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BCHE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown. Nonlinearity of the plots may be explained in terms of compressibility changes or rate-limiting changes. To distinguish between these two possibilities, enzyme phosphorylation by diisopropylfluorophosphate (DFP) in the presence of substrate (BzSCh) under pressure was studied. There was no pressure dependence of volume changes for DFP binding or for phosphorylation of either wild-type or D70G. Analysis of the pressure dependence for steady-state hydrolysis of substrates, and for phosphorylation by DFP provided evidence that no enzyme compressibility changes occurred during the catalyzed reactions. Thus, the nonlinear pressure dependence of substrate hydrolysis reflects changes in the rate-limiting step with pressure. Change in rate-determining step occurred at a pressure of 100 MPa for hydrolysis of BzCh by wild-type and at 75 MPa for D70G. For hydrolysis of BzSCh the change occurred at higher pressures because k(2) << k(3) at atmospheric pressure for this substrate. Elementary volume change contributions upon initial binding, productive binding, acylation and deacylation were calculated from the pressure differentiation of kinetic constants. This analysis shed light on the molecular events taking place along the hydrolysis pathways of BzCh and BzSCh by wild-type BCHE and the D70G mutant. In addition, volume change differences between wild-type and D70G provided new evidence that residue D70 in the peripheral site controls hydration of the active site gorge and the dynamics of the water molecule network during catalysis. Finally, a steady-state kinetic study of the oxyanion hole mutant (G117H) showed that substitution of the ethereal sulfur for oxygen in the substrate alters the final adjustment of substrate in the active site and stabilization of the acylation transition state.
ESTHER : Masson_2004_Eur.J.Biochem_271_1980
PubMedSearch : Masson_2004_Eur.J.Biochem_271_1980
PubMedID: 15128307

Title : Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate - Masson_2004_Eur.J.Biochem_271_220
Author(s) : Masson P , Goldstein BN , Debouzy JC , Froment MT , Lockridge O , Schopfer LM
Ref : European Journal of Biochemistry , 271 :220 , 2004
Abstract : Steady-state kinetics for the hydrolysis of benzoylcholine (BzCh) and benzoylthiocholine (BzSCh) by wild-type human butyrylcholinesterase (BuChE) and by the peripheral anionic site mutant D70G were compared. kcat/Km for the hydrolysis of BzSCh was 17-fold and 32-fold lower than that for hydrolysis of BzCh by wild-type and D70G, respectively. The rate-limiting step for hydrolysis of BzCh was deacylation, whereas acylation was rate-limiting for hydrolysis of BzSCh. Wild-type enzyme and the D70G mutant were found to reach steady-state velocity slowly with BzCh as the substrate. At pH 6, the approach to steady-state for both enzymes consisted of a mono-exponential acceleration upon which a set of damped oscillations was superimposed. From pH 7 to 8.5, the approach to steady-state consisted of a simple exponential acceleration. The damped oscillations were analyzed by both a numerical approximation and simulation based on a theoretical model. BuChE-catalyzed hydrolysis of the thiocholine analogue of BzCh showed neither lags nor oscillations, under the same conditions. The frequency and amplitude of the damped oscillations decreased as the BzCh concentration increased. The apparent induction time for the exponential portion of the lag was calculated from the envelope of the damped oscillations or from the smooth lag. Wild-type BuChE showed a hyperbolic increase in induction time as the BzCh concentration increased (tau max = 210 s at pH 6.0). However, the induction time for D70G was constant over the whole range of BzCh concentrations (tau max = 60 s at pH 6.0). Thus, the induction time does not conform to a simple hysteretic model in which there is a slow conformational transition of the enzyme from an inactive form E to an active form E'. No pH-dependence of the induction time was found between pH 6.0 and 8.5 in sodium phosphate buffers of various concentrations (from 1 mm to 1 m). However, increasing the pH tended to abolish the oscillations (increase the damping factor). This effect was more pronounced for D70G than for wild-type. Although the lyotropic properties of phosphate change from chaotropic at pH 6.0 to kosmotropic at pH > 8.0, no effect of phosphate concentration on the oscillations was noticed at the different pH values, suggesting that the oscillations are not related to a pH-dependent Hofmeister effect of phosphate ions. Simulation and theoretical analysis of the oscillatory behaviour of the approach to the steady-state for BuChE led us to propose a model for the hysteresis of BuChE with BzCh. In this model, the substrate-free enzyme is present as an equilibrium mixture of two forms, E and E'. Substrate binds to E and E', but only Epsilon'S makes products. It is proposed that oscillations originate from a time-dependent change in the local concentration, solvation and/or conformation of substrate in the bulk solution. 1H-NMR measurements provided evidence for a slow equilibrium between two BzCh conformers. Binding of the conformationally preferred substrate conformer leads to products.
ESTHER : Masson_2004_Eur.J.Biochem_271_220
PubMedSearch : Masson_2004_Eur.J.Biochem_271_220
PubMedID: 14686935

Title : Hysteresis in butyrylcholinesterase catalysis. -
Author(s) : Masson P , Froment MT , Nachon F , Schopfer LM
Ref : Cholinergic Mechanisms, CRC Press :637 , 2004
PubMedID:

Title : Capillary zone electrophoresis detects unwanted cholinesterase-bound hidden ligands that alter enzyme conformational stability. -
Author(s) : Rochu D , Renault F , Bon C , Masson P
Ref : Cholinergic Mechanisms, CRC Press :675 , 2004
PubMedID:

Title : X-Ray structures of native and soman-aged human butyrylcholinesterase. -
Author(s) : Nachon F , Nicolet Y , Masson P , Lockridge O , Fontecilla-Camps JC
Ref : Cholinergic Mechanisms, CRC Press :165 , 2004
PubMedID:

Title : Poster (93) How do the crystal structures of human butyrylcholinesterase compare to torpedo californica acetylcholinesterases structures? -
Author(s) : Nachon F , Nicolet Y , Masson P , Lockridge O , Fontecilla-Camps JC
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :369 , 2004
PubMedID:

Title : The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron-scattering study - Gabel_2004_Biophys.J_86_3152
Author(s) : Gabel F , Weik M , Doctor BP , Saxena A , Fournier D , Brochier L , Renault F , Masson P , Silman I , Zaccai G
Ref : Biophysical Journal , 86 :3152 , 2004
Abstract : A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.
ESTHER : Gabel_2004_Biophys.J_86_3152
PubMedSearch : Gabel_2004_Biophys.J_86_3152
PubMedID: 15111428

Title : Hysteretic behavior of butyrylcholinesterase: Kinetic curiosity or catalytically and physiologicaly significant? -
Author(s) : Masson P , Froment MT , Nachon F , Lockridge O , Schopfer LM
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :191 , 2004
PubMedID:

Title : Poster (20) Hysteretic behavior of butyrylcholinesterase : kinetic curiosity or catalytically and physiologically significant? -
Author(s) : Masson P , Froment MT , Nachon F , Lockridge O , Schopfer LM
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :331 , 2004
PubMedID:

Title : Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products - Nicolet_2003_J.Biol.Chem_278_41141
Author(s) : Nicolet Y , Lockridge O , Masson P , Fontecilla-Camps JC , Nachon F
Ref : Journal of Biological Chemistry , 278 :41141 , 2003
Abstract : Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase, involved in the hydrolysis of the neurotransmitter acetylcholine, and butyrylcholinesterase of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge, raising the question of how substrate and products enter and leave. Human butyrylcholinesterase (BChE) has attracted attention because it can hydrolyze toxic esters such as cocaine or scavenge organophosphorus pesticides and nerve agents. Here we report the crystal structures of several recombinant truncated human BChE complexes and conjugates and provide a description for mechanistically relevant non-productive substrate and product binding. As expected, the structure of BChE is similar to a previously published theoretical model of this enzyme and to the structure of Torpedo acetylcholinesterase. The main difference between the experimentally determined BChE structure and its model is found at the acyl binding pocket that is significantly bigger than expected. An electron density peak close to the catalytic Ser(198) has been modeled as bound butyrate.
ESTHER : Nicolet_2003_J.Biol.Chem_278_41141
PubMedSearch : Nicolet_2003_J.Biol.Chem_278_41141
PubMedID: 12869558
Gene_locus related to this paper: human-BCHE

Title : High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine - Masson_2003_Eur.J.Biochem_270_315
Author(s) : Masson P , Nachon F , Bartels CF , Froment MT , Ribes F , Matthews C , Lockridge O
Ref : European Journal of Biochemistry , 270 :315 , 2003
Abstract : Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as alpha-chymotrypsin. Enzymes of this type are generally considered to be inactive at pH values below 5, because the histidine member of the catalytic triad becomes protonated. We have found that butyrylcholinesterase retains activity at pH
ESTHER : Masson_2003_Eur.J.Biochem_270_315
PubMedSearch : Masson_2003_Eur.J.Biochem_270_315
PubMedID: 12605682

Title : Photoreversible inhibition of cholinesterases: catalytic serine-labeled caged butyrylcholinesterase - Loudwig_2003_Chembiochem_4_762
Author(s) : Loudwig S , Nicolet Y , Masson P , Fontecilla-Camps JC , Bon S , Nachon F , Goeldner M
Ref : Chembiochem , 4 :762 , 2003
Abstract : The photoregulation of the catalytic activity of butyrylcholinesterase (BChE) was investigated by treating the enzyme with a newly developed carbamylating reagent, N-methyl-N-(2-nitrophenyl)carbamoyl chloride, which has proved to be an efficient photoremovable alcohol-protecting group. BChE was efficiently inhibited in a time- and concentration-dependent manner, and the enzyme could be protected against inhibition by active-site-specific ligands (that is, tacrine). The inactivation kinetics showed a biphasic character, which can be analyzed as the result of the existence of two conformational states in solution. Pseudo-irreversible inactivation of BChE, which results from catalytic serine carbamylation, was suggested by recovery of the enzyme activity after dilution and was demonstrated by X-ray crystallography. Remarkably, the 3D structure of the carbamylated BChE conjugate showed a nonambiguous carbamylation of the catalytic serine residue as the only chemical modification on the protein. The photoreversibility of the enzyme inactivation was analyzed by irradiating the inactivated enzyme at 365 nm and was shown to reach completion in some conditions. The efficient and specific "caging" of BChE, together with the availability of carbamylated BChE crystals, will offer a unique possibility to study the catalytic properties of this enzyme by kinetic crystallography after cryophotolytic uncaging of the enzyme conjugate crystals.
ESTHER : Loudwig_2003_Chembiochem_4_762
PubMedSearch : Loudwig_2003_Chembiochem_4_762
PubMedID: 12898628

Title : Substrate activation in acetylcholinesterase induced by low pH or mutation in the pi-cation subsite - Masson_2002_Biochim.Biophys.Acta_1594_313
Author(s) : Masson P , Schopfer LM , Bartels CF , Froment MT , Ribes F , Nachon F , Lockridge O
Ref : Biochimica & Biophysica Acta , 1594 :313 , 2002
Abstract : Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the pi-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the pi-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.
ESTHER : Masson_2002_Biochim.Biophys.Acta_1594_313
PubMedSearch : Masson_2002_Biochim.Biophys.Acta_1594_313
PubMedID: 11904227

Title : Pressure and heat inactivation of recombinant human acetylcholinesterase. Importance of residue E202 for enzyme stability - Clery-Barraud_2002_Eur.J.Biochem_269_4297
Author(s) : Clery-Barraud C , Ordentlich A , Grosfeld H , Shafferman A , Masson P
Ref : European Journal of Biochemistry , 269 :4297 , 2002
Abstract : The effects of pressure on structure and activity of recombinant human acetylcholinesterase (rHuAChE) were investigated up to a pressure of 300 MPa using gel electrophoresis under elevated hydrostatic pressure, fluorescence of bound 8-anilinonaphthalene-1-sulfonate (ANS) and activity measurements following exposure to high pressure. Study of wild-type enzyme and three single mutants (D74N, E202Q, E450A) and one sextuple mutant (E84Q/E292A/D349N/E358Q/E389Q/D390N) showed that pressure exerts a differential action on wild-type rHuAChE and its mutants, allowing estimation of the contribution of carboxylic amino acid side-chains to enzyme stability. Mutation of negatively charged residues D74 and E202 by polar side-chains strengthened heat or pressure stability. The mutation E450A and the sextuple mutation caused destabilization of the enzyme to pressure. Thermal inactivation data on mutants showed that all of them were stabilized against temperature. In conclusion, pressure and thermal stability of mutants provided evidence that the residue E202 is a determinant of structural and functional stability of HuAChE.
ESTHER : Clery-Barraud_2002_Eur.J.Biochem_269_4297
PubMedSearch : Clery-Barraud_2002_Eur.J.Biochem_269_4297
PubMedID: 12199708

Title : Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization - Nachon_2002_Eur.J.Biochem_269_630
Author(s) : Nachon F , Nicolet Y , Viguie N , Masson P , Fontecilla-Camps JC , Lockridge O
Ref : European Journal of Biochemistry , 269 :630 , 2002
Abstract : Human butyrylcholinesterase (BChE; EC 3.1.1.8) is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine, organophosphorus pesticides and nerve agents. The relative contribution of each N-linked glycan for the solubility, the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. The purified recombinant BChE showed catalytic properties similar to those of the native enzyme. Tetragonal crystals suitable for X-ray crystallography studies were obtained; they were improved by recrystallization and found to diffract to 2.0 A resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a = b = 154.7 A, c = 124.9 A, giving a Vm of 2.73 A3 per Da (estimated 60% solvent) for a single molecule of recombinant BChE in the asymmetric unit. The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general.
ESTHER : Nachon_2002_Eur.J.Biochem_269_630
PubMedSearch : Nachon_2002_Eur.J.Biochem_269_630
PubMedID: 11856322

Title : Butyrylcholinesterase-catalyzed hydrolysis of N-methylindoxyl acetate: analysis of volume changes upon reaction and hysteretic behavior - Masson_2002_Biochim.Biophys.Acta_1597_229
Author(s) : Masson P , Froment MT , Fort S , Ribes F , Bec N , Balny C , Schopfer LM
Ref : Biochimica & Biophysica Acta , 1597 :229 , 2002
Abstract : Hydrolysis of the neutral substrate N-methylindoxyl acetate (NMIA) by wild-type human butyrylcholinesterase (BuChE) and peripheral site mutants (D70G, Y332A, D70G/Y332A) was found to follow the Michaelis-Menten kinetics. K(m) was 0.14 mM for wild-type, and 0.07-0.16 mM for D70G, Y332A and D70G/Y332A, indicating that the peripheral site is not involved in NMIA binding. The values of k(cat) were of the same order for all enzymes: 12,000-18,000 min(-1). Volume changes upon substrate binding (-DeltaV(K(m))) and the activation volumes (DeltaV++(k(cat)) associated with hydrolysis of NMIA were calculated from the pressure dependence of the catalytic constants. Values of -DeltaV(K(m)) indicate that NMIA binds to an aromatic residue, presumed to be W82, the active site binding locus. Binding is accompanied by a release of water molecules from the gorge. Residue 70 controls the number of water molecules that are released upon substrate binding. The values of DeltaV++(k(cat)), which are positive for wild-type and faintly positive for D70G, clearly indicate that the catalytic steps are accompanied by re-entry of water into the gorge. Results support the premise that residue D70 is involved in the conformational stabilization of the active site gorge and in control of its hydration. A slow transient, preceding the steady state, was seen on a time scale of several minutes. The induction time rapidly increased with NMIA concentration to reach a limit at substrate saturation. Much shorter induction times (<1 min) were seen for hydrolysis of benzoylcholine (BzCh) by wild-type BuChE and for hydrolysis of butyrylthiocholine (BuSCh) by the active site mutants E197Q and E197Q/G117H. This slow transient was interpreted in terms of hysteresis without kinetic cooperativity. The hysteretic behavior of BuChE results from a slow conformational equilibrium between two enzyme states E and E'. NMIA binds only to the primed form E'. Kosmotropic salts and hydrostatic pressure were found to shift the equilibrium toward E'. The E-->E' transition is accompanied by a negative activation volume (DeltaV++(0)= -45+/-10 ml/mol), and the E' form is more compact than E. Hydration water in the gorge of E' appears to be more structured than in the unprimed form.
ESTHER : Masson_2002_Biochim.Biophys.Acta_1597_229
PubMedSearch : Masson_2002_Biochim.Biophys.Acta_1597_229
PubMedID: 12044901

Title : Concentration-dependent reversible activation-inhibition of human butyrylcholinesterase by tetraethylammonium ion - Stojan_2002_Eur.J.Biochem_269_1154
Author(s) : Stojan J , Golicnik M , Froment MT , Estour F , Masson P
Ref : European Journal of Biochemistry , 269 :1154 , 2002
Abstract : Tetraalkylammonium (TAA) salts are well known reversible inhibitors of cholinesterases. However, at concentrations around 10 mm, they have been found to activate the hydrolysis of positively charged substrates, catalyzed by wild-type human butyrylcholinesterase (EC 3.1.1.8) [Erdoes, E.G., Foldes, F.F., Zsigmond, E.K., Baart, N. & Zwartz, J.A. (1958) Science 128, 92]. The present study was undertaken to determine whether the peripheral anionic site (PAS) of human BCHE (Y332, D70) and/or the catalytic substrate binding site (CS) (W82, A328) are involved in this phenomenon. For this purpose, the kinetics of butyrylthiocholine (BTC) hydrolysis by wild-type human BCHE, by selected mutants and by horse BCHE was carried out at 25 degreeC and pH 7.0 in the presence of tetraethylammonium (TEA). It appears that human enzymes with more intact structure of the PAS show more prominent activation phenomenon. The following explanation has been put forward: TEA competes with the substrate at the peripheral site thus inhibiting the substrate hydrolysis at the CS. As the inhibition by TEA is less effective than the substrate inhibition itself, it mimics activation. At the concentrations around 40 mm, well within the range of TEA competition at both substrate binding sites, it lowers the activity of all tested enzymes.
ESTHER : Stojan_2002_Eur.J.Biochem_269_1154
PubMedSearch : Stojan_2002_Eur.J.Biochem_269_1154
PubMedID: 11856351

Title : Detection of unwanted protein-bound ligands by capillary zone electrophoresis: the case of hidden ligands that stabilize cholinesterase conformation - Rochu_2002_Electrophoresis_23_930
Author(s) : Rochu D , Renault F , Masson P
Ref : Electrophoresis , 23 :930 , 2002
Abstract : Detection, identification and characterization of compounds present in purified proteins and biopharmaceuticals are of central interest. As well as chemical remedies, proteins of pharmacological interest have to exhibit their nakedness to become therapeutic drugs. Cholinesterases (ChE) are enzymes of major importance for detoxification of poisonous esters. Likewise, ChE are characterized by the high catalytic efficiency of an active site positioned at the bottom of a deep gorge. The gorge can be partially or fully occupied by ligands, i.e., substrates and inhibitors that are currently used in affinity chromatography purification steps. Accordingly, a suitable method allowing to analyse the presence of unwanted ligands and its influence on the functional conformation and stability of these enzymes was essential. We have developed CZE approaches for that purpose. The factors causing discrepancies between data for thermal unfolding of ChE by electrophoretic and by calorimetric methods were investigated. The presence of unwanted hidden ligands bound to purified enzymes was first demonstrated. The incidence of these ligands was discussed. Altogether, our results raised several questions concerning the real conformation of the native state of enzymes. Finally, CZE was proved to be a pertinent tool to validate the conformity of purified enzymes to a status of biopharmaceutical.
ESTHER : Rochu_2002_Electrophoresis_23_930
PubMedSearch : Rochu_2002_Electrophoresis_23_930
PubMedID: 11920879

Title : Thermal stability of acetylcholinesterase from Bungarus fasciatus venom as investigated by capillary electrophoresis - Rochu_2001_Biochim.Biophys.Acta_1545_216
Author(s) : Rochu D , Georges C , Repiton J , Viguie N , Saliou B , Bon C , Masson P
Ref : Biochimica & Biophysica Acta , 1545 :216 , 2001
Abstract : Previous studies on the conformation of the monomeric acetylcholinesterase (AChE) from the krait (Bungarus fasciatus) venom showed that the protein possesses a large permanent dipole moment. These studies predicted that thermal irreversible denaturation must occur via partially unfolded states. The thermal stability of Bungarus AChE was determined using capillary electrophoresis (CE) with optimized conditions. Runs performed at convenient temperature scanning rates provided evidence for an irreversible denaturation process according to the Lumry and Eyring model. The mid-transition temperature, T(m), and the effective enthalpy change, DeltaH(m) were determined at different pH. The temperature dependence of the free energy, DeltaG, of Bungarus AChE unfolding was drawn using values of T(m), DeltaH(m) and DeltaC(p) determined by CE. The thermodynamic parameters for the thermal denaturation of the monomeric snake enzyme were compared with those of different dimeric and tetrameric ChEs. It was shown that the changes in the ratio of DeltaH(cal/)DeltaH(vH) and DeltaC(p) reflect the oligomerization state of these proteins. All these results indicate that wild-type monomeric Bungarus AChE is a stable enzyme under standard conditions. However, designed mutants of this enzyme capable of degrading organophosphates have to be engineered to enhance their thermostability.
ESTHER : Rochu_2001_Biochim.Biophys.Acta_1545_216
PubMedSearch : Rochu_2001_Biochim.Biophys.Acta_1545_216
PubMedID: 11342047

Title : Pressure- and heat-induced inactivation of butyrylcholinesterase: evidence for multiple intermediates and the remnant inactivation process - Weingand-Ziade_2001_Biochem.J_356_487
Author(s) : Weingand-Ziade A , Ribes F , Renault F , Masson P
Ref : Biochemical Journal , 356 :487 , 2001
Abstract : The inactivation process of native (N) human butyrylcholinesterase (BCHE) by pressure and/or heat was found to be multi-step. It led to irreversible formation of an active intermediate (I) state and a denatured state. This series-inactivation process was described by expanding the Lumry-Eyring [Lumry, R. and Eyring, H. (1954) J. Phys. Chem. 58, 110-120] model. The intermediate state (I) was found to have a K(m) identical with that of the native state and a turnover rate (k(cat)) twofold higher than that of the native state with butyrylthiocholine as the substrate. The increased catalytic efficiency (k(cat)/K(m)) of I can be explained by a conformational change in the active-site gorge and/or restructuring of the water-molecule network in the active-site pocket, making the catalytic steps faster. However, a pressure/heat-induced covalent modification of native BCHE, affecting the catalytic machinery, cannot be ruled out. The inactivation process of BCHE induced by the combined action of pressure and heat was found to continue after interruption of pressure/temperature treatment. This secondary inactivation process was termed 'remnant inactivation'. We hypothesized that N and I were in equilibrium with populated metastable N' and I' states. The N' and I' states can either return to the active forms, N and I, or develop into inactive forms, N(')(in) and I(')(in). Both active N' and I' intermediate states displayed different rates of remnant inactivation depending on the pressure and temperature pretreatments and on the storage temperature. A first-order deactivation model describing the kinetics of the remnant inactivation of BCHE is proposed.
ESTHER : Weingand-Ziade_2001_Biochem.J_356_487
PubMedSearch : Weingand-Ziade_2001_Biochem.J_356_487
PubMedID: 11368776

Title : Dual effect of high electric field in capillary electrophoresis study of the conformational stability of Bungarus fasciatus acetylcholinesterase - Rochu_2001_J.Chromatogr.A_910_347
Author(s) : Rochu D , Pernet T , Renault F , Bon C , Masson P
Ref : Journal of Chromatography A , 910 :347 , 2001
Abstract : The effect of high electric field in capillary zone electrophoresis (CZE) was evaluated for the study of the thermally induced unfolding of Bungarus fasciatus acetylcholinesterase. This monomer enzyme is characterised by two interdependent uncommon structural features, the asymmetrical distribution of charged residues and a relatively low thermal denaturation temperature. Both traits were presumed to interfere in the thermal unfolding of this enzyme as investigated by CZE. This paper analyses the effect of high electric field on the behaviour of the enzyme native state. It is shown that increasing the applied field causes denaturation-like transition of the enzyme at a current power which does not induce excessive Joule heating in the capillary. The susceptibility to electric field of proteins like cholinesterases, with charge distribution anisotropy, large permanent dipole moment and notable molecular flexibility associated with moderate thermal stability, was subsequently discussed.
ESTHER : Rochu_2001_J.Chromatogr.A_910_347
PubMedSearch : Rochu_2001_J.Chromatogr.A_910_347
PubMedID: 11261729

Title : Effects of mutations of active site residues and amino acids interacting with the Omega loop on substrate activation of butyrylcholinesterase - Masson_2001_Biochim.Biophys.Acta_1544_166
Author(s) : Masson P , Xie W , Froment MT , Lockridge O
Ref : Biochimica & Biophysica Acta , 1544 :166 , 2001
Abstract : The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.
ESTHER : Masson_2001_Biochim.Biophys.Acta_1544_166
PubMedSearch : Masson_2001_Biochim.Biophys.Acta_1544_166
PubMedID: 11341926
Gene_locus related to this paper: human-BCHE

Title : The active site of human paraoxonase (PON1) - Josse_2001_J.Appl.Toxicol_21 Suppl 1_S7
Author(s) : Josse D , Lockridge O , Xie W , Bartels CF , Schopfer LM , Masson P
Ref : J Appl Toxicol , 21 Suppl 1 :S7 , 2001
Abstract : Ideally we would like to treat people exposed to nerve agents with an enzyme that rapidly destroys nerve agents. The enzymes considered for such a role include human butyrylcholinesterase (BChE), acetylcholinesterase (AChE), carboxylesterase and paraoxonase (PON1). Success has been achieved in endowing BChE with the ability to hydrolyze organophosphates. The G117H mutant of BCHE hydrolyzes sarin and VX, whereas the double mutant G117H/E197Q hydrolyzes soman (Millard et al. Biochemistry 1995; 34: 15925-15933; 1998; 37: 237-247). However, the rates of organophosphate hydrolysis are slow and a faster organophosphate hydrolase is being sought. Native PON1 hydrolyzes paraoxon with a catalytic efficiency, of 2.4 x 10(6) M(-1) x min(-1), and our goal is to improve the organophosphate hydrolase activity of PON1. To achieve this we need to identify the amino acids in the active site of PON1. Using site-directed mutagenesis and expression in human 293T cells, we have identified the following eight amino acids as being essential to PON1 activity: W280, H114, H133, H154, H242, H284, E52 and D53. Fluorescence of PON1 complexed to terbium ion shows that at least one tryptophan is close to the calcium binding site.
ESTHER : Josse_2001_J.Appl.Toxicol_21 Suppl 1_S7
PubMedSearch : Josse_2001_J.Appl.Toxicol_21 Suppl 1_S7
PubMedID: 11920913

Title : Pesticides and susceptible populations: people with butyrylcholinesterase genetic variants may be at risk - Lockridge_2000_Neurotoxicol_21_113
Author(s) : Lockridge O , Masson P
Ref : Neurotoxicology , 21 :113 , 2000
Abstract : Butyrylcholinesterase (BChE) scavenges low doses of organophosphorus (for example, paraoxon) and carbamate pesticides (for example, carbaryl) and in this way protects people from the toxic effects of these poisons. The protective role of BChE is demonstrated by the finding that pesticide applicators can have reduced BChE activity with no clinical signs of poisoning. The question has arisen whether people with genetic variants of BChE are less protected. Seventy-six percent of the population is homozygous for wild-type BChE, while 24% carry at least one genetic variant allele. Most genetic variants of BChE have reduced activity. The clinically most important variant is atypical (D70G) BChE because people with this variant have 2 hours of apnea after receiving a dose of succinylcholine that is intended to paralyze muscles for 3-5 minutes. In test tube experiments the atypical variant reacts more slowly with all positively charged compounds (for example physostigmine, echothiophate). This leaves more toxin available for reaction with acetylcholinesterase in nerve synapses and predicts that people with atypical BChE will be less protected. Variants with low activity, such as silent BChE, are predicted to be at increased risk from organophosphorus pesticides based on experiments in monkeys and rodents where injection of purified BChE protected animals from the toxic effects of nerve agents. More studies are needed to strengthen the hypothesis that people with genetic variants of BChE are at higher risk of intoxication from pesticides.
ESTHER : Lockridge_2000_Neurotoxicol_21_113
PubMedSearch : Lockridge_2000_Neurotoxicol_21_113
PubMedID: 10794391

Title : Identification of residues essential for human paraoxonase (PON1) arylesterase\/organophosphatase activities - Josse_1999_Biochemistry_38_2816
Author(s) : Josse D , Xie W , Renault F , Rochu D , Schopfer LM , Masson P , Lockridge O
Ref : Biochemistry , 38 :2816 , 1999
Abstract : Human serum paraoxonase (PON1) is a calcium-dependent organophosphatase. To identify residues essential for PON1 activity, we adopted complementary approaches based on chemical modification and site-directed mutagenesis. To detect 45Ca2+ binding to native and chemically modified PON1, we performed nondenaturating gel electrophoresis. The environment of calcium-binding sites was probed using the Ca2+ analogue, terbium. Tb3+ binds to calcium-binding sites as shown by displacement of 45Ca2+ by Tb3+. Binding of Tb3+ is accompanied by a complete loss of enzyme activity. PON1 chemical modification with the Trp-selective reagent, N-bromosuccinimide, and the Asp/Glu-selective, dicyclohexylcarbodiimide, established that Trp and Asp/Glu residues are components of the PON1 active center and calcium-binding sites. Additional evidence for the presence of a Trp residue in the PON1 calcium-binding sites was a characteristic fluorescence emission at 545 nm from the PON1-Tb3+ complex and abolishment of that fluorescence upon modification by N-bromosuccinimide. The importance of aromatic/hydrophobic character of the residue 280 was demonstrated by site-directed mutagenesis: the W280F mutant was fully active while the W280A and W280L mutants had markedly reduced activity. Twelve amino acids among conserved His and Asp/Glu residues were found essential for PON1 arylesterase and organophosphatase activities: H114, H133, H154, H242, H284, D53, D168, D182, D268, D278, E52, and E194. Finally, the cysteines constituting the PON1 disulfide bond (C41 and C352) were essential, but the glycan chains linked to Asn 252 and 323 were not essential for PON1 secretion and activity.
ESTHER : Josse_1999_Biochemistry_38_2816
PubMedSearch : Josse_1999_Biochemistry_38_2816
PubMedID: 10052953

Title : Differential effect of pressure and temperature on the catalytic behaviour of wild-type human butyrylcholinesterase and its D70G mutant - Weingand-Ziade_1999_Eur.J.Biochem_264_327
Author(s) : Weingand-Ziade A , Renault F , Masson P
Ref : European Journal of Biochemistry , 264 :327 , 1999
Abstract : The combined action of temperature (10-35 degrees C) and pressure (0. 001-2 kbar) on the catalytic activity of wild-type human butyrylcholinesterase (BCHE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BCHE. Results showed a break in Arrhenius plots of wild-type BCHE (at Tt approximately 22 degrees C) whatever the pressure (dTt/dP = 1.6 +/- 1.5 degrees C.kbar-1), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature-induced conformational change of the wild-type BCHE which did not occur for the D70G mutant. For the wild-type BCHE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild-type BCHE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 degrees C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BCHE, and is involved in regulation of the catalytic activity as a function of temperature.
ESTHER : Weingand-Ziade_1999_Eur.J.Biochem_264_327
PubMedSearch : Weingand-Ziade_1999_Eur.J.Biochem_264_327
PubMedID: 10491076

Title : Human serum paraoxonase (PON1): identification of essential amino acid residues by group-selective labelling and site-directed mutagenesis - Josse_1999_Chem.Biol.Interact_119-120_71
Author(s) : Josse D , Xie W , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 119-120 :71 , 1999
Abstract : Human serum paraoxonase/arylesterase (PON1, EC 3.1.8.1.) is a calcium-dependent enzyme which hydrolyzes a wide variety of organophosphates, including paraoxon, DFP, sarin and soman. Although the 3-D structure of PON has not yet been determined and its sequence shows no similarity with any other crystallized proteins, we undertook to identify some of its essential amino acid residues by two complementary approaches: group-specific labelling and site-directed mutagenesis. Group-specific labelling studies, performed on the purified native enzyme, indicated that one or more Trp, His and Asp/Glu are potentially important residues for PON activity. Based on these results, we identified some of these residues, conserved in the sequenced mammalian PON1, by site-directed mutagenesis. PON1 mutants were transiently expressed in 293T cells. The catalytic constants k(cat) and Km (relative to k(cat) and Km of the wild-type) determined with four different substrates (phenylacetate, paraoxon, diazoxon, chlorpyrifos oxon), were not significantly changed for the following mutants: W193A, W201A, W253A, H160N, H245N, H250N, H347N, E32A, E48A, D88A, D107A, D121A, D273A. By contrast, k(cat) was less than 1% for eight mutants: W280A, H114N, H133N, H154N, H242N, H284N, E52A and D53A. The essential amino acid residues identified in this work could be part of the PON1 active site, acting either as calcium ligands (E52 and D53?) or as substrate binding (W280?) or nucleophilic (His residues?) sites. However, we cannot rule out that the effects of mutations on catalytic properties resulted from a remote conformational change and/or misfolding of mutant proteins.
ESTHER : Josse_1999_Chem.Biol.Interact_119-120_71
PubMedSearch : Josse_1999_Chem.Biol.Interact_119-120_71
PubMedID: 10421440

Title : Tryptophan residue(s) as major components of the human serum paraoxonase active site - Josse_1999_Chem.Biol.Interact_119-120_79
Author(s) : Josse D , Xie W , Masson P , Schopfer LM , Lockridge O
Ref : Chemico-Biological Interactions , 119-120 :79 , 1999
Abstract : Serum paraoxonase (PON1, EC 3.1.8.1.) is a high density lipid- (HDL)-associated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. The kinetic parameters k(cat) and Km (relative to k(cat) and Km of the wild-type), determined with four substrates (phenylacetate, paraoxon, diazoxon and chlorpyrifosoxon) were less than 1, and more than 100% for the W280A and W280F mutant enzymes, respectively. These results indicated that the aromatic/hydrophobic character of the amino acid in position 280 is essential for PON1 activity. In this study, we investigated whether this aromatic residue is in the PON1 active site. Group-specific labelling studies with N-bromosuccinimide, an oxidative agent of tryptophan, strongly suggested that one or several Trp could be in the active site of PON1 but we could not conclude either on the specificity of the labelling reaction or on the number of oxidized Trp. However, although PON activity was not altered by the hydrophilic tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl chloride (NBC), it was significantly reduced by the p-nitrophenylacetate analog 2-acetoxy-5-nitrobenzyl chloride (ANBC), whose hydrolysis by PON1 generated NBC in the active site. Moreover, since at least one calcium ion is present in the PON catalytic site, we attempted to probe the metal local environment using the calcium analog terbium. The luminescence spectrum of the PON terbium complex exhibited an emission peak at 545 nm characteristic of an aromatic residue (Trp and/or Tyr)-terbium interaction. In conclusion, both the results obtained with the mechanism-based inhibitor of PON1 (ANBC) and the calcium-binding site luminescent probe terbium support the hypothesis of the presence of at least one Trp residue in the PON1 active site. Trp residue(s) may be involved in the binding of aromatic substrates.
ESTHER : Josse_1999_Chem.Biol.Interact_119-120_79
PubMedSearch : Josse_1999_Chem.Biol.Interact_119-120_79
PubMedID: 10421441

Title : Structural and hydration changes in the active site gorge of phosporhylated butyrylcholinesterase accompanying the aging process - Masson_1999_Chem.Biol.Interact_119-120_17
Author(s) : Masson P , Fortier PL , Albaret C , Clery C , Guerra P , Lockridge O
Ref : Chemico-Biological Interactions , 119-120 :17 , 1999
Abstract : Wild-type (wt) butyrylcholinesterase (BuChE) and the E197D and D70G mutants were inhibited by diisopropylfluorophosphate (DFP) or soman under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process of DFP-phosphorylated enzymes (diisopropylphosphoryl-BuChE (DIP-BuChE)) was investigated. Hydrostatic pressure strongly increased the rate of aging of wt enzyme. The activation volumes (deltaV*) for the dealkylation reaction was -150 ml/mol for DIP-wtBuChE. On the other hand, pressure had little effect on the aging of the DIP-E197D mutant and no effect on the DIP-D70G mutant, indicating that the transition state of the aging reaction (dealkylation of an isoproxy chain) was associated with an extended conformation/hydration change in wtBuChE but not in mutants. The rate of aging decreased with osmotic pressure, supporting the idea that water is important for stabilizing the transition state. Molecular dynamics simulations were performed on the wtDIP adduct to relate the kinetic data to hydration changes in the enzyme active site gorge. The pH dependence of the melting temperature (Tm) of native and soman-wtBuChE, as determined by differential scanning calorimetry (DSC), indicated that the stabilization energy of aged BuChE is mainly due to the salt bridge between protonated H438 and PO-, with pK(H438) = 8.3. Electrophoresis under high pressure up to 2.5 kbar showed that aged wtBuChE did not undergo pressure-induced molten globule transition unlike the native enzyme. This transition was not seen for the mutant enzymes, indicating that mutants are resistant to the penetration of water into their structure. Our results support the conclusion that D70 and E197 are major residues for the water/H-bond network dynamics in the active site gorge of BuChE, both residues acting like valves. In mutant enzymes, mutated residues function like check valves: forced penetration of water in the gorge is difficult, release of water is facilitated.
ESTHER : Masson_1999_Chem.Biol.Interact_119-120_17
PubMedSearch : Masson_1999_Chem.Biol.Interact_119-120_17
PubMedID: 10421435

Title : Hydration change during the aging of phosphorylated human butyrylcholinesterase: importance of residues aspartate-70 and glutamate-197 in the water network as probed by hydrostatic and osmotic pressures - Masson_1999_Biochem.J_343_361
Author(s) : Masson P , Clery C , Guerra P , Redslob A , Albaret C , Fortier PL
Ref : Biochemical Journal , 343 :361 , 1999
Abstract : Wild-type human butyrylcholinesterase (BCHE) and Glu-197-->Asp and Asp-70-->Gly mutants (E197D and D70G respectively) were inhibited by di-isopropyl phosphorofluoridate under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process (dealkylation of an isopropyl chain) of phosphorylated enzymes [di-isopropylated (DIP)-BCHE] was investigated. Hydrostatic pressure markedly increased the rate of aging of wild-type enzyme. The average activation volume (DeltaV( not equal)) for the dealkylation reaction was -170 ml/mol for DIP wild-type BCHE. On the other hand, hydrostatic pressure had little effect on the aging of the DIP mutants (DeltaV( not equal)=-2.6 ml/mol for E197D and -2 ml/mol for D70G), suggesting that the transition state of the aging process was associated with an extended hydration and conformational change in wild-type BCHE, but not in the mutants. The rate of aging of wild-type and mutant enzymes decreased with osmotic pressure, allowing very large positive osmotic activation volumes (DeltaV not equal osm) to be estimated, thus probing the participation of water in the aging process. Molecular dynamics simulations performed on the active-site gorge of the wild-type DIP adduct showed that the isopropyl chain involved in aging was highly solvated, supporting the idea that water is important for stabilizing the transition state of the dealkylation reaction. Wild-type BCHE was inhibited by soman (pinacolyl methylphosphonofluoridate). Electrophoresis performed under high pressure [up to 2.5 kbar (1 bar=10(5) Pa)] showed that the soman-aged enzyme did not pass through a pressure-induced, molten-globule transition, unlike the native wild-type enzyme. Likewise, this transition was not seen for the native E197D and D70G mutants, indicating that these mutants are resistant to the penetration of water into their structure. The stability energetics of native and soman-aged wild-type BCHE were determined by differential scanning calorimetry. The pH-dependence of the midpoint transition temperature of endotherms indicated that the high difference in stabilization energy between aged and native BCHE (DeltaDeltaG=23.7 kJ/mol at pH 8.0) is mainly due to the salt bridge between protonated His-438 and PO(-), with pK(His-438)=8.3. A molecular dynamics simulation on the MIP adduct showed that there is no water molecule around the ion pair. The 'hydrostatic versus osmotic pressure' approach probed the importance of water in aging, and also revealed that Asp-70 and Glu-197 are the major residues controlling both the dynamics and the structural organization of the water/hydrogen-bond network in the active-site gorge of BCHE. In wild-type BCHE both residues function like valves, whereas in the mutant enzymes the water network is slack, and residues Gly-70 and Asp-197 function like check valves, i.e. forced penetration of water into the gorge is not easily achieved, thereby facilitating the release of water.
ESTHER : Masson_1999_Biochem.J_343_361
PubMedSearch : Masson_1999_Biochem.J_343_361
PubMedID: 10510301

Title : Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant - Levitsky_1999_Biochim.Biophys.Acta_1429_422
Author(s) : Levitsky V , Xie W , Froment MT , Lockridge O , Masson P
Ref : Biochimica & Biophysica Acta , 1429 :422 , 1999
Abstract : Wild-type human butyrylcholinesterase (BCHE) has a non-Michaelian behaviour showing substrate activation with butyrylthiocholine (BTC) as the substrate. The D70G mutant has a catalytic constant identical to that of the wild-type enzyme, but a 10-fold lower affinity for BTC compared to wild-type enzyme, and it does not exhibit activation by excess BTC under conventional conditions. In the present work it was found that addition of polyols or sugars changed the kinetic behaviour of the D70G mutant with BTC. In the presence of 40% sucrose, the D70G mutant enzyme displayed marked activation by excess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 were studied. Mutant Y332F had a behaviour similar to that of wild-type BCHE, whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activation. The behavior of wild-type, Y332F, Y332A and Y332A/D70G did not change in the presence of high concentrations of sugar. Substrate activation has been explained by binding of a second substrate molecule in the peripheral site at D70. The D70G mutant should be incapable of substrate activation, if D70 were the only residue involved in substrate activation. The ability of the D70G mutant to display substrate activation by medium engineering suggests that other residues are involved in initial substrate binding and activation by excess substrate. Osmolyte-induced change in conformation and/or hydration status of Y332 and other solvent-exposed residues may account for the non-Michaelian behaviour of the D70G mutant.
ESTHER : Levitsky_1999_Biochim.Biophys.Acta_1429_422
PubMedSearch : Levitsky_1999_Biochim.Biophys.Acta_1429_422
PubMedID: 9989227

Title : Interaction between the peripheral site residues of human butyrylcholinesterase, D70 and Y332, in binding and hydrolysis of substrates - Masson_1999_Biochim.Biophys.Acta_1433_281
Author(s) : Masson P , Xie W , Froment MT , Levitsky V , Fortier PL , Albaret C , Lockridge O
Ref : Biochimica & Biophysica Acta , 1433 :281 , 1999
Abstract : Human butyrylcholinesterase displays substrate activation with positively charged butyrylthiocholine (BTC) as the substrate. Peripheral anionic site (PAS) residues D70 and Y332 appear to be involved in the initial binding of charged substrates and in activation control. To determine the contribution of PAS residues to binding and hydrolysis of quaternary substrates and activation control, the single mutants D70G/Y and Y332F/A/D and the double mutants Y332A/D70G and Y332D/D70Y were studied. Steady-state hydrolysis of the charged substrates, BTC and succinyldithiocholine, and the neutral ester o-nitrophenyl butyrate was measured. In addition, inhibition of wild-type and mutant enzymes by tetramethylammonium was investigated, at low concentrations of BTC. Single and double mutants of D70 and Y332 showed little or no substrate activation, suggesting that both residues were important for activation control. The effects of double mutations on D70 and Y332 were complex. Double-mutant cycle analysis provided evidence for interaction between these residues. The category of interaction (either synergistic, additive, partially additive or antagonistic) was found to depend on the nature of the substrate and on measured binding or kinetic parameters. This complexity reflects both the cross-talk between residues involved in the sequential formation of productive Michaelian complexes and the effect of peripheral site residues on catalysis. It is concluded that double mutations on the PAS induce a conformational change in the active site gorge of butyrylcholinesterase that can alter both substrate binding and enzyme acylation.
ESTHER : Masson_1999_Biochim.Biophys.Acta_1433_281
PubMedSearch : Masson_1999_Biochim.Biophys.Acta_1433_281
PubMedID: 10446378

Title : [Huperzine a: an acetylcholinesterase inhibitor with high pharmacological potential] - Pilotaz_1999_Ann.Pharm.Fr_57_363
Author(s) : Pilotaz F , Masson P
Ref : Ann Pharm Fr , 57 :363 , 1999
Abstract : Huperzine A is an alkaloid isolated from a chinese club-moss. The molecule is a potent and selective inhibitor of acetylcholinesterase. Several pharmacological and clinical studies showed that huperzine A improves the mnesic capacity and cognitive functions. Huperzine A was also found to be a neuroprotective agent. This molecule which possesses a high pharmacological potential is under clinical evaluation for the palliative treatment of Alzheimer's disease. In addition, its use in the pretreatment of poisoning by organophosphorous nerve agents could be another indication.
ESTHER : Pilotaz_1999_Ann.Pharm.Fr_57_363
PubMedSearch : Pilotaz_1999_Ann.Pharm.Fr_57_363
PubMedID: 10520506

Title : Two invertebrate acetylcholinesterases show activation followed by inhibition with substrate concentration - Marcel_1998_Biochem.J_329_329
Author(s) : Marcel V , Palacios LG , Pertuy C , Masson P , Fournier D
Ref : Biochemical Journal , 329 :329 , 1998
Abstract : In vertebrates there are two cholinesterases, with differences in catalytic behaviour with respect to substrate concentration: butyrylcholinesterase displays an increased activity at low substrate concentrations, whereas acetylcholinesterase displays inhibition by excess substrate. In two invertebrates, Drosophila melanogaster and Caenorhabditis elegans, we found cholinesterases that showed both kinetic complexities: substrate activation at low substrate concentrations followed by inhibition at higher concentrations. These triphasic kinetics can be explained by the presence of two enzymes with different kinetic behaviours or more probably by the existence of a single enzyme regulated by the substrate concentration.
ESTHER : Marcel_1998_Biochem.J_329_329
PubMedSearch : Marcel_1998_Biochem.J_329_329
PubMedID: 9425116

Title : Enzymes hydrolyzing organophosphates as potential catalytic scavengers against organophosphate poisoning - Masson_1998_J.Physiol.Paris_92_357
Author(s) : Masson P , Josse D , Lockridge O , Viguie N , Taupin C , Buhler C
Ref : Journal de Physiologie (Paris) , 92 :357 , 1998
Abstract : Enzymes hydrolyzing organophosphates could be used as catalytic scavengers for treatment of organophosphate poisoning and for decontamination. Two organophosphorus hydrolases (OPH) were selected: the Flavobacterium sp/Pseudomonas diminuta phosphotriesterase (PTE) and human paraoxonase (HuPON). Genes encoding these enzymes were cloned and functional recombinant enzymes expressed. PTE was expressed in E. coli. Natural HuPON was purified from human plasma; recombinant HuPON was expressed in human embryonic kidney 293 T cells. Although HuPON displays interesting catalytic properties, a site-directed mutagenesis program was undertaken to improve its catalytic efficiency. PTE has high efficiency in hydrolysis of organophosphates, including nerve agents. PTE injected in rat has a half-life of 100 min. However, to overcome pharmacokinetic problems of injected OPH and/or immunological incompatibility, the model enzyme (recombinant PTE) was immobilized onto a hollow-fiber reactor. This reactor designed for extracorporeal blood circulation is under experimentation for post-exposure detoxification.
ESTHER : Masson_1998_J.Physiol.Paris_92_357
PubMedSearch : Masson_1998_J.Physiol.Paris_92_357
PubMedID: 9789837

Title : Mechanical Aspects of the Phosphotriesterase Activity of Human Butyrylcholinesterase G117H Mutant -
Author(s) : Albaret C , Masson P , Broomfield CA , Kaim LE , Fortier PL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :399 , 1998
PubMedID:

Title : Structural Changes in the Active Site Gorge of Phosphylated Cholinesterase Accompanying the Aging Process -
Author(s) : Masson P , Clery C , Guerra P , Fortier PL , Albaret C , Lockridge O
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :419 , 1998
PubMedID:

Title : Purification, molecular characterization and catalytic properties of a Pseudomonas fluorescens enzyme having cholinesterase-like activity - Rochu_1998_Biochim.Biophys.Acta_1385_126
Author(s) : Rochu D , Rothlisberger C , Taupin C , Renault F , Gagnon J , Masson P
Ref : Biochimica & Biophysica Acta , 1385 :126 , 1998
Abstract : An enzyme with a cholinesterase (ChE) activity, produced by Pseudomonas fluorescens, was purified to homogeneity in a three-step procedure. Analysis by non-denaturing and SDS-PAGE, and by isoelectric focusing, indicated that the enzyme was a monomer of 43 kDa, with a pI of 6.1. The N-terminal sequence, AEPLKAVGAGEGQLDIVAWPGYIEA, showed some similarities with proteins of the ChE family and a strong similarity with a protein from Escherichia coli with unknown structure and function. Cholinesterase activity at pH 7.0 and 25 degreesC was maximum with propionylthiocholine as substrate (kcat,app=670 min-1), followed by acetylthiocholine, and significantly lower with butyrylthiocholine. Catalytic specificity (kcat/Km) was the same for propionylthiocholine and acetylthiocholine, but was two orders of magnitude lower for butyrylthiocholine. Kinetics of thiocholine ester hydrolysis showed inhibition by excess substrate which was ascribed to binding of a second substrate molecule, leading to non-productive ternary complex (Km=35 microM, KSS=0.49 mM with propionylthiocholine). There was low or no reactivity with organophosphates and carbamates. The enzyme inhibited by echothiophate (kII=0.44x102 M-1 min-1) was not reactivated by pralidoxime methiodide. However, the P. fluorescens enzyme had affinity for procainamide and decamethonium, two reversible ChE inhibitors used as affinity chromatography ligand and eluant, respectively. Although similarity of the N-terminal amino acid sequence of the enzyme with an internal sequence of ChEs is weak, its catalytic activity towards thiocholine esters, and its affinity for positively charged ligands supports the contention that this enzyme may belong to the ChE family. However, we cannot rule out that the enzyme belongs to another structural family of proteins having cholinesterase-like properties. The reaction of the enzyme with organophosphates suggests that it is a serine esterase, and currently this enzyme may be termed as having a cholinesterase-like activity.
ESTHER : Rochu_1998_Biochim.Biophys.Acta_1385_126
PubMedSearch : Rochu_1998_Biochim.Biophys.Acta_1385_126
PubMedID: 9630567

Title : Poster: Mechanical Aspects of the Phosphotriesterase Activity of Human Butyrylcholinesterase G117H Mutant -
Author(s) : Albaret C , Masson P , Broomfield CA , Kaim LE , Fortier PL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :434 , 1998
PubMedID:

Title : Stability of the Quaternary Structure of Butyrylcholinesterase Subjected to Ultrasound or Hydrostatic Pressure -
Author(s) : Froment MT , Clery C , Weingand-Ziade A , Masson P
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :436 , 1998
PubMedID:

Title : Absence of a protective effect of the oxime 2-PAM toward paraoxon-poisoned honey bees: acetylcholinesterase reactivation not at fault - Polyzou_1998_Toxicol.Appl.Pharmacol_152_184
Author(s) : Polyzou A , Froment MT , Masson P , Belzunces LP
Ref : Toxicol Appl Pharmacol , 152 :184 , 1998
Abstract : We investigated the failure of 2-PAM to protect honey bees against poisoning with paraoxon. The protective effect of the oxime 2-PAM against inhibition of acetylcholinesterase (AChE) by paraoxon was estimated in vitro and in vivo and was correlated with the mortality of paraoxon-treated bees. In vitro, 2-PAM protected 90% of AChE activity in the presence of paraoxon and reactivated more than 90% of inhibited AChE. Minor soluble and major membrane-bound forms of bee AChE presented about similar extents of reactivation, but the first order rate constant of reactivation (kobs) of the soluble form is threefold higher than that of the membrane-bound form. However, this difference did not significantly influence the reactivation kinetics of total AChE; the constant kobs of the membrane-bound form reflected that of total AChE. The linear kinetic profile of total AChE reactivation supported the conclusion that there was a single population of reactivatable species. The bimolecular rate constant of reactivation (kr), the dephosphorylation rate constant (k2), and the dissociation constant (Kd) were 646 M-1.min-1, 0.84 min-1 and 1. 30 mM, respectively. In vivo, administration of 2-PAM, after paraoxon exposure, induced a complete protection of AChE activity, but did not elicit any significant effect on mortality in paraoxon-treated bees. The inefficiency of 2-PAM to antagonize paraoxon-induced mortality was not changed by the administration of 2-PAM in pretreatment-therapy and in therapy treatments. These results indicated that the mortality of paraoxon-poisoned honey bees was not due to a lack of AChE reactivation.
ESTHER : Polyzou_1998_Toxicol.Appl.Pharmacol_152_184
PubMedSearch : Polyzou_1998_Toxicol.Appl.Pharmacol_152_184
PubMedID: 9772214

Title : A putative kinetic model for substrate metabolisation by Drosophila acetylcholinesterase - Stojan_1998_FEBS.Lett_440_85
Author(s) : Stojan J , Marcel V , Estrada-Mondaca S , Klaebe A , Masson P , Fournier D
Ref : FEBS Letters , 440 :85 , 1998
Abstract : Insect acetylcholinesterase, an enzyme whose catalytic site is located at the bottom of a gorge, can metabolise its substrate in a wide range of concentrations (from 1 microM to 200 mM) since it is activated at low substrate concentrations. It also presents inhibition at high substrate concentrations. Among the various rival kinetic models tested to analyse the kinetic behaviour of the enzyme, the simplest able to explain all the experimental data suggests that there are two sites for substrate molecules on the protein. Binding on the catalytic site located at the bottom of the gorge seems to be irreversible, suggesting that each molecule of substrate which enters the active site gorge is metabolised. Reversible binding at the peripheral site of the free enzyme has high affinity (2 microM), suggesting that this binding increases the probability of the substrate entering the active site gorge. Peripheral site occupation decreases the entrance rate constant of the second substrate molecule to the catalytic site and strongly affects the catalytic activity of the enzyme. On the other hand, catalytic site occupation lowers the affinity of the peripheral site for the substrate (34 mM). These effects between the two sites result both in apparent activation at low substrate concentration and in general inhibition at high substrate concentration.
ESTHER : Stojan_1998_FEBS.Lett_440_85
PubMedSearch : Stojan_1998_FEBS.Lett_440_85
PubMedID: 9862431

Title : Shielding of Acetylcholinesterase does not Result in the Protection of Honey Bee against Poisoning by Organophosphates -
Author(s) : Polyzou A , Froment MT , Masson P , Belzunces LP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :552 , 1998
PubMedID:

Title : Resistance of butyrylcholinesterase to inactivation by ultrasound: effects of ultrasound on catalytic activity and subunit association - Froment_1998_Biochim.Biophys.Acta_1387_53
Author(s) : Froment MT , Lockridge O , Masson P
Ref : Biochimica & Biophysica Acta , 1387 :53 , 1998
Abstract : The effects of 20 kHz ultrasound on catalytic activity and structure of the tetramer of wild-type human butyrylcholinesterase (BChE) from plasma and recombinant D70G mutant enzyme were studied at constant temperature. Effects on catalytic properties of both enzymes were investigated by kinetic analysis under ultrasound irradiation using a neutral substrate (o-nitrophenylbutyrate), a positively charged substrate (butyrylthiocholine), and a negatively charged substrate (aspirin). Effects on structure of highly purified wild-type BChE were followed by gel electrophoresis and activity measurements at Vmax after ultrasound treatment. Unlike hydrostatic pressure, mild ultrasound had moderate effects on catalytic parameters of BChE-catalyzed hydrolysis of substrates. For both wild-type and D70G, Km increased slightly with butyrylthiocholine and o-nitrophenylbutyrate under ultrasound irradiation, suggesting that these effects of ultrasound were not due to the periodic variation of pressure but rather to shear forces that took off substrate from the peripheral site and altered diffusion to the active site. By contrast, affinity of the D70G mutant for aspirin slightly increased with ultrasound power, suggesting that ultrasound-induced microstreaming unmasked peripheral residues involved in recognition and initial binding of the negatively charged substrate. Results support the contention that Km is a composite affinity constant, including dissociation constant of the first encounter enzyme-substrate complex on the peripheral site. Small changes in catalytic activity may have resulted from ultrasound-induced subtle conformational changes altering the active site reactivity. Short ultrasound irradiation induced a faint transient enzyme activation, but prolonged irradiation caused partial dissociation of the tetrameric enzyme and irreversible inactivation. Partial dissociation was related to enzyme microheterogeneity, i.e., nicked (C-terminal segment depleted) tetramers were less stable than native tetramers. The resistance of the native tetramer to ultrasound-induced dissociation was ascribed to the existence of an aromatic amino acid array on the apolar side of the C-terminal helical segment of subunits, the four subunits being held together in a four-helix bundle containing the aromatic zipper motifs. Aromatic/aromatic interactions between the four helical segments are thought to be enhanced by ultrasound-generated pressure.
ESTHER : Froment_1998_Biochim.Biophys.Acta_1387_53
PubMedSearch : Froment_1998_Biochim.Biophys.Acta_1387_53
PubMedID: 9748500

Title : Butyrylcholinesterase-catalysed hydrolysis of aspirin, a negatively charged ester, and aspirin-related neutral esters - Masson_1998_Biochim.Biophys.Acta_1387_41
Author(s) : Masson P , Froment MT , Fortier PL , Visicchio JE , Bartels CF , Lockridge O
Ref : Biochimica & Biophysica Acta , 1387 :41 , 1998
Abstract : Although aspirin (acetylsalicylic acid) is negatively charged, it is hydrolysed by butyrylcholinesterase (BCHE). Catalytic parameters were determined in 100 mM Tris buffer, pH 7.4, in the presence and absence of metal cations. The presence of Ca2+ or Mg2+ (<100 mM) in buffer did not change the Km, but accelerated the rate of hydrolysis of aspirin by wild-type or D70G mutant BCHE by 5-fold. Turnover numbers were of the order of 5000-12000 min-1 for the wild-type enzyme and the D70G and D70K enzymes in 100 mM Tris, pH 7.4, containing 50 mM CaCl2 at 25 degreesC; Km values were 6 mM for wild-type, 16 mM for D70G and 38 mM for D70K. People with 'atypical' BCHE have the D70G mutation. The apparent inhibition seen at high aspirin concentration was not due to inhibition by excess substrate but to spontaneous hydrolysis of aspirin, causing inhibition by salicylate. The wild-type and D70G enzymes were competitively inhibited by salicylic acid; the D70K enzyme showed a complex parabolic inhibition, suggesting multiple binding. The effect of salicylate was substrate-dependent, the D70K mutant being activated by salicylate with butyrylthiocholine as substrate. Km value for wild-type enzyme was lower than for D70 mutants, suggesting that residue 70 located at the rim of the active site gorge was not the major site for the initial encounter aspirin-BCHE complex. On the other hand, the virtual absence of affinity of the W82A mutant for aspirin indicated that W82 was the major residue involved in formation of the Michaelis complex. Molecular modelling of aspirin binding to BCHE indicated perpendicular interactions between the aromatic rings of W82 and aspirin. Kinetic study of BCHE-catalysed hydrolysis of different acetyl esters showed that the rate limiting step was acetylation. The bimolecular rate constants for hydrolysis of aspirin by wild-type, D70G and D70K enzymes were found to be close to 1x106 M-1 min-1. These results support the contention that the electrostatic steering due to the negative electrostatic field of the enzyme plays a role in substrate binding, but plays no role in the catalytic steps, i.e. in the enzyme acetylation.
ESTHER : Masson_1998_Biochim.Biophys.Acta_1387_41
PubMedSearch : Masson_1998_Biochim.Biophys.Acta_1387_41
PubMedID: 9748494

Title : A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase - Lockridge_1997_Biochemistry_36_786
Author(s) : Lockridge O , Blong RM , Masson P , Froment MT , Millard CB , Broomfield CA
Ref : Biochemistry , 36 :786 , 1997
Abstract : The G117H mutant of human butyrylcholinesterase (EC 3.1.1.8) was expressed in Chinese hamster ovary cells. Substitution of Gly 117 with His to make the G117H mutant endowed butyrylcholinesterase with the ability to catalyze the hydrolysis of organophosphate esters. G117H was still able to hydrolyze butyrylthiocholine, benzoylcholine, and o-nitrophenyl butyrate, but in addition it had acquired the ability to hydrolyze the antiglaucoma drug echothiophate and the pesticide paraoxon. Wild-type butyrylcholinesterase was irreversibly inhibited by echothiophate and paraoxon, but G117H regained 100% activity within 2-3 min following reaction with these compounds. On a polyacrylamide gel, the same bands that stained for activity with butyrylthiocholine also stained for activity with echothiophate. G117H is the only enzyme known that hydrolyzes echothiophate. Diethoxyphosphorylated G117H aged with a half-time of 5.5 h, a rate 600 times slower than the rate of hydrolysis. Echothiophate and paraoxon were hydrolyzed with the same kcat of 0.75 min-1. This calculates to a rate acceleration of 100,000-fold for hydrolysis of echothiophate and paraoxon by the G117H mutant compared to the nonenzymatic rate.
ESTHER : Lockridge_1997_Biochemistry_36_786
PubMedSearch : Lockridge_1997_Biochemistry_36_786
PubMedID: 9020776

Title : Importance of aspartate-70 in organophosphate inhibition, oxime re-activation and aging of human butyrylcholinesterase - Masson_1997_Biochem.J_325_53
Author(s) : Masson P , Froment MT , Bartels CF , Lockridge O
Ref : Biochemical Journal , 325 :53 , 1997
Abstract : Asp-70 is the defining amino acid in the peripheral anionic site of human butyrylcholinesterase (BCHE), whereas acetylcholinesterase has several additional amino acids, the most important one being Trp-277 (Trp-279 in Torpedo AChE). We studied mutants D70G, D70K and A277W to evaluate the role of Asp-70 and Trp-277 in reactions with organophosphates. We found that Asp-70 was important for binding positively charged echothiophate, but not neutral paraoxon and iso-OMPA. Asp-70 was also important for binding of positively charged pralidoxime (2-PAM) and for activation of re-activation by excess 2-PAM. Excess 2-PAM had an effect similar to substrate activation, suggesting the binding of 2 mol of 2-PAM to wild-type but not to the D70G mutant. A surprising result was that Asp-70 was important for irreversible aging, the D70G mutant having a 3- and 8-fold lower rate of aging for paraoxon-inhibited and di-isopropyl fluorophosphate-inhibited BCHE. Mutants of Asp-70 had the same rate constants for phosphorylation and re-activation by 2-PAM as wild-type. The A277W mutant behaved like wild-type in all assays. Our results predict that people with the atypical (D70G) variant of BCHE will be more sensitive to the toxic effects of echothiophate, but will be equally sensitive to paraoxon and di-isopropyl fluorophosphate. People with the D70G mutation will be resistant to re-activation of their inhibited BCHE by 2-PAM, but this will be offset by the lower rate of irreversible aging of inhibited BCHE, allowing some regeneration by spontaneous hydrolysis.
ESTHER : Masson_1997_Biochem.J_325_53
PubMedSearch : Masson_1997_Biochem.J_325_53
PubMedID: 9224629

Title : Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase - Masson_1997_Biochemistry_36_2266
Author(s) : Masson P , Legrand P , Bartels CF , Froment MT , Schopfer LM , Lockridge O
Ref : Biochemistry , 36 :2266 , 1997
Abstract : The atypical variant of human butyrylcholinesterase has Gly in place of Asp 70. Patients with this D70G mutation respond abnormally to the muscle relaxant succinyldicholine, experiencing hours of apnea rather than the intended 3 min. Asp 70 is at the rim of the active site gorge 12 A from the active site Ser 198. An unanswered question in the literature is why the atypical variant has a 10-fold increase in Km for compounds with a single positive charge but a 100-fold increase in Km for compounds with two positive charges. We mutated residues Asp 70, Trp 82, Trp 231, Glu 197, and Tyr 332 and expressed mutant enzymes in mammalian cells. Steady-state kinetic parameters for hydrolysis of butyrylthiocholine, benzoylcholine, succinyldithiocholine, and o-nitrophenyl butyrate were determined. The wild type and the D70G mutant had identical k(cat) values for all substrates. Molecular modeling and molecular dynamics suggested that succinyldicholine could bind in two consecutive orientations in the active site gorge; formation of one complex caused a conformational change in the omega loop involving Asp 70 and Trp 82. We propose the formation of three enzyme-substrate intermediates preceding the acyl-enzyme intermediate; kinetic data support this contention. Substrates with a single positive charge interact with Asp 70 just once, whereas substrates with two positive charges, for example succinyldithiocholine, interact with Asp 70 in two complexes, thus explaining the 10- and 100-fold increases in Km in the D70G mutant.
ESTHER : Masson_1997_Biochemistry_36_2266
PubMedSearch : Masson_1997_Biochemistry_36_2266
PubMedID: 9047329

Title : Aging of di-isopropyl-phosphorylated human butyrylcholinesterase - Masson_1997_Biochem.J_327_601
Author(s) : Masson P , Fortier PL , Albaret C , Froment MT , Bartels CF , Lockridge O
Ref : Biochemical Journal , 327 ( Pt 2) :601 , 1997
Abstract : Organophosphate-inhibited cholinesterases can be reactivated by nucleophilic compounds. Sometimes phosphylated (phosphorylated or phosphonylated) cholinesterases become progressively refractory to reactivation; this can result from different reactions. The most frequent process, termed 'aging', involves the dealkylation of an alkoxy group on the phosphyl moiety through a carbocation mechanism. In attempting to determine the amino acid residues involved in the aging of butyrylcholinesterase (BuChE), the human BuChE gene was mutated at several positions corresponding to residues located at the rim of the active site gorge and in the vicinity of the active site. Mutant enzymes were expressed in Chinese hamster ovary cells. Wild-type BuChE and mutants were inhibited by di-isopropylfluorophosphate at pH 8.0 and 25 degrees C. Di-isopropyl-phosphorylated enzymes were incubated with the nucleophilic oxime 2-pyridine aldoxime methiodide and their reactivatability was determined. Reactivatability was expressed by the first-order rate constant of aging and/or the half-life of aging (t12). The t12 was found to be of the order of 60 min for wild-type BuChE. Mutations on Glu-197 increased t12 60-fold. Mutation W82A increased t12 13-fold. Mutation D70G increased t12 8-fold. Mutations in the vicinity of the active site serine residue had either moderate or no effect on aging; t12 was doubled for F329C and F329A, increased only 4-fold for the double mutant A328G+F329S, and no change was observed for the A328G mutant, indicating that the isopropoxy chain to be dealkylated does not directly interact with Ala-328 and Phe-329. These results were interpreted by molecular modelling of di-isopropylphosphorylated wild-type and mutant enzymes. Molecular dynamics simulations indicated that the isopropyl chain that is lost interacted with Trp-82, suggesting that Trp-82 has a role in stabilizing the carbonium ion that is released in the dealkylation step. This study emphasized the important role of the Glu-197 carboxylate in stabilizing the developing carbocation, and the allosteric control of the dealkylation reaction by Asp-70. Indeed, although Asp-70 does not interact with the phosphoryl moiety, mutation D70G affects the rate of aging. This indirect control was interpreted in terms of change in the conformational state of Trp-82 owing to internal motions of the Omega loop (Cys-65-Cys-92) in the mutant enzyme.
ESTHER : Masson_1997_Biochem.J_327_601
PubMedSearch : Masson_1997_Biochem.J_327_601
PubMedID: 9359435

Title : Combined pressure\/heat-induced inactivation of butyrylcholinesterase - Weingand-Ziade_1997_Biochim.Biophys.Acta_1340_245
Author(s) : Weingand-Ziade A , Renault F , Masson P
Ref : Biochimica & Biophysica Acta , 1340 :245 , 1997
Abstract : The combined effects of pressure and temperature on the activity of butyrylcholinesterase (BuChE) were investigated in the pressure range from 10(-3) to 5 kbar and temperature range from -10 degrees C to 70 degrees C. Inactivation of the enzyme showed a complex dependence on pressure and temperature. Under moderate pressures (1-3 kbar) at temperatures 40-65 degrees C BuChE was resistant to heat inactivation; under other conditions of pressure and temperature, the action of both parameters was synergistic and caused inactivation. Results allowed to construct a pressure-temperature kinetic phase diagram for the enzyme inactivation. The elliptic diagram for the irreversible transition active-->inactive BuChE as a function of both pressure and temperature has a positive angular coefficient. This indicates that pressure acts as a stabilizer of BuChE against heat denaturation.
ESTHER : Weingand-Ziade_1997_Biochim.Biophys.Acta_1340_245
PubMedSearch : Weingand-Ziade_1997_Biochim.Biophys.Acta_1340_245
PubMedID: 9252111

Title : Asp7O in the peripheral anionic site of human butyrylcholinesterase - Masson_1996_Eur.J.Biochem_235_36
Author(s) : Masson P , Froment MT , Bartels CF , Lockridge O
Ref : European Journal of Biochemistry , 235 :36 , 1996
Abstract : The goal of this work was to determine what amino acids at the mouth of the active-site gorge are important for the function of human butyrylcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G283D were expressed in human embryonal kidney cells and the secreted enzymes were assayed by steady-state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 was mutated to G, the same mutation as in the naturally occurring atypical butyrylcholinesterase, the affinity for positively charged substrates and positively charged inhibitors decreased 5-30-fold. The D70G mutant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine. Thus, though kcat was the same for wild-type and D70G mutant, being 24000 min(-1) at low butyrylthiocholine concentrations (0.01-0.1 mM), it failed to increase for the D70G mutant at 40 mM butyrylthiocholine, whereas it increased threefold for wild type. The D70G mutant was more sensitive to changes in salt concentration, its catalytic rate decreasing more than that of the wild type. The D70G mutant appeared to have a greater surface negative charge than wild type suggesting that the D70G mutant had a conformation different from that of the wild type. That D70 affects the function of butyrylcholinesterase, together with its location at the mouth of the active-site gorge, supports the hypothesis that D70 is a component of the peripheral anionic site of butyrylcholinesterase. Mutants containing aromatic amino acids at the mouth of the gorge had increased binding affinity for propidium and fasciculin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyrylcholinesterase.
ESTHER : Masson_1996_Eur.J.Biochem_235_36
PubMedSearch : Masson_1996_Eur.J.Biochem_235_36
PubMedID: 8631355

Title : Subunit Association and Stabilization of Butyrylcholinesterase (BChE) -
Author(s) : Blong RM , Masson P , Lockridge O
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :129 , 1995
PubMedID:

Title : Substrate dependence of amiloride- and soman-induced conformation changes of butyrylcholinesterase as evidenced by high-pressure perturbation - Clery_1995_Biochim.Biophys.Acta_1250_19
Author(s) : Clery C , Heiber-Langer I , Channac L , David L , Balny C , Masson P
Ref : Biochimica & Biophysica Acta , 1250 :19 , 1995
Abstract : Previous results on butyrylcholinesterase-catalyzed hydrolysis of o-nitrophenylbutyrate in the presence of soman, an irreversible inhibitor of cholinesterases, suggested that reversible binding of soman preceding enzyme phophonylation induced a new enzyme conformational state (E'). The purpose of the present study was to determine whether this effect depends on soman itself or is dependent on the presence and nature of substrate or ligand. First, we examined the effect of amiloride, a reversible cholinesterase effector, upon the butyrylcholinesterase-catalyzed hydrolysis of nitrophenyl esters. The effect of amiloride was found to be dependent on the position ortho or para of the substrate nitro group: amiloride acts as a non-linear reversible activator of p-nitrophenyl ester hydrolysis and as a non-linear reversible inhibitor of o-nitrophenyl ester hydrolysis. Second, the effect of amiloride upon hydrolysis of o/p-nitrophenylbutyrate was also studied under perturbing conditions, i.e., as a function of pressure (1-1600 bar) in the presence and absence of soman. Results show that the effect of reversible soman binding on butyrylcholinesterase activity in the presence of amiloride depends on the position of the substrate nitro group and amiloride concentration. Molecular modelling suggests that the presence of amiloride determines the orientation of ortho- and para-nitrophenyl esters in the active-site. gorge. The nitro group of o-nitrophenylbutyrate interacts with the oxyanion hole via hydrogen bonds and its phenyl ring interacts with amiloride whose heterocycle faces Trp-82. The nitro group of p-nitrophenylbutyrate does not interact with the oxyanion hole but points towards Tyr-332; the phenyl ring of p-nitrophenylbutyrate interacts with amiloride but there is no steric constraint on the acyl chain. Thus, the network of interactions in ternary complexes is tighter with o-nitrophenylbutryate as the substrate. There is no evidence for the existence of amiloride and/or soman-induced E' state when p-nitrophenylbutyrate is the substrate. On the other hand, reversible binding of amiloride and/or soman induces new active conformational states that may be either binary (or ternary) enzyme-ligand complex or new free enzyme conformation resulting from long-lived ligand-induced enzyme conformational change when o-nitrophenylbutyrate is the substrate. These ligand-induced states are stabilized by high pressure.
ESTHER : Clery_1995_Biochim.Biophys.Acta_1250_19
PubMedSearch : Clery_1995_Biochim.Biophys.Acta_1250_19
PubMedID: 7612649

Title : Pressure Effects on Structure and Activity of Cholinesterase -
Author(s) : Masson P , Clery C
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :113 , 1995
PubMedID:

Title : Denaturation of Recombinant Human Acetylcholinesterase -
Author(s) : Lebleu M , Clery C , Masson P , Reuveny S , Marcus D , Velan B , Shafferman A
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :131 , 1995
PubMedID:

Title : Insect Acetylcholinesterase and Resistance to Insecticides -
Author(s) : Fournier D , Maturano M , Gagnoux L , Ziliani P , Pertuy C , Pralavorio M , Bride JM , Elmarbouh L , Klaebe A , Masson P
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :149 , 1995
PubMedID:

Title : Pressure-induced molten globule state of cholinesterase - Clery_1995_FEBS.Lett_370_212
Author(s) : Clery C , Renault F , Masson P
Ref : FEBS Letters , 370 :212 , 1995
Abstract : The denaturing effect of pressure on the structure of human butyrylcholinesterase was examined by gel electrophoresis under pressure and by 8-anilino-1-naphthalene sulfonate (ANS) binding. It was found that the fluorescence intensity of bound ANS is increased by pressure between 0.5 and 1.5 kbar and that the hydrodynamic volume of the enzyme swells when pressures around 1.5 kbar are applied. These findings indicate that pressure denaturation of butyrylcholinesterase is a multi-step process and that the observed transient pressure-denatured states have characteristics of molten globules.
ESTHER : Clery_1995_FEBS.Lett_370_212
PubMedSearch : Clery_1995_FEBS.Lett_370_212
PubMedID: 7656979

Title : Kinetics of butyrylcholinesterase in reversed micelles under high pressure - Clery_1995_Biochim.Biophys.Acta_1253_85
Author(s) : Clery C , Bec N , Balny C , Mozhaev VV , Masson P
Ref : Biochimica & Biophysica Acta , 1253 :85 , 1995
Abstract : The combined effects of high pressure and reversed micelles have been studied to modulate the catalytic behaviour of butyrylcholinesterase. The purpose of this study was to determine whether the conformational plasticity of the enzyme is altered by entrapment in reversed micelles. The presence of soman, an irreversible inhibitor of cholinesterase was used to bring to the fore a possible modification of the enzyme behaviour in this system under pressure. Results show differences between enzyme in conventional medium and in reversed micelles regarding the mechanism of BCHE catalyzed hydrolysis of acetylthiocholine. In both systems, the enzyme displays a non-Michaelian behaviour with this substrate. In conventional medium the kinetics is multiphasic with an activation phase followed by an inhibition phase at high concentration. In reversed micelles there is inhibition by excess substrate but the activation phase is missing. This behaviour may be the result of a change of the enzyme conformational plasticity when is entrapped in reversed micelles.
ESTHER : Clery_1995_Biochim.Biophys.Acta_1253_85
PubMedSearch : Clery_1995_Biochim.Biophys.Acta_1253_85
PubMedID: 7492604

Title : Peripheral Anionic Site of Wild-Type and Mutant Human Butyrylcholinesterase -
Author(s) : Masson P , Froment MT , Bartels CF , Lockridge O
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :230 , 1995
PubMedID:

Title : Raman spectroscopic study of conjugates of butyrylcholinesterase with organophosphates - Aslanian_1995_Biochim.Biophys.Acta_1249_37
Author(s) : Aslanian D , Grof P , Renault F , Masson P
Ref : Biochimica & Biophysica Acta , 1249 :37 , 1995
Abstract : Raman spectra of human butyrylcholinesterase (BCHE; E.C. 3.1.1.8) were analyzed in the native state and after conjugation with organophosphates (soman, DFP and paraoxon). The secondary structure of the native BCHE in Tris-HCl buffer (pH 7.5), determined from analysis of the amide I polypeptide vibration band, indicates 47% alpha-helices, 26% beta-sheets, 16% turns and 12% undefined structure. We obtained the same values for paraoxon-phosphorylated BCHE, but 39% helical structure, 31% beta-sheets, 17% turns and 13% undefined structure for 'aged' DFP-BCHE conjugates and 36% helical structure, 34% beta-sheets, 20% turns and 10% undefined structure for 'aged' soman-BCHE conjugates. The approximately 10% decrease of alpha-helical structure observed upon phosphorylation by DFP and phosphonylation by soman, probably corresponds to the 'aging' process, which does not take place in the case of paraoxon. Considerable differences have been observed between native, paraoxon inhibited and 'aged' BCHE in aromatic ring vibrations, suggesting that the dealkylation of organophosphate conjugates modifies the environment or the interactions of aromatic amino-acid residues. In the aliphatic side chains an increase of the number of gauche configurations has been observed in 'aged' DFP-BCHE and soman-BCHE
ESTHER : Aslanian_1995_Biochim.Biophys.Acta_1249_37
PubMedSearch : Aslanian_1995_Biochim.Biophys.Acta_1249_37
PubMedID: 7766682

Title : Endogenous butyrylcholinesterase in SV40 transformed cell lines: COS-1, COS-7, MRC-5 SV40, and WI-38 VA13 - Kris_1994_In.Vitro.Cell.Dev.Biol.Animal_10_680
Author(s) : Kris M , Jbilo O , Bartels CF , Masson P , Rhode S , Lockridge O
Ref : In Vitro Cellular & Developmental Biology Animal , 10 :680 , 1994
Abstract : Comparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.
ESTHER : Kris_1994_In.Vitro.Cell.Dev.Biol.Animal_10_680
PubMedSearch : Kris_1994_In.Vitro.Cell.Dev.Biol.Animal_10_680
PubMedID: 7842168

Title : Mutation His322Asn in human acetylcholinesterase does not alter electrophoretic and catalytic properties of the erythrocyte enzyme - Masson_1994_Blood_83_3003
Author(s) : Masson P , Froment MT , Sorenson RC , Bartels CF , Lockridge O
Ref : Blood , 83 :3003 , 1994
Abstract : The YT blood group antigen is located on human red blood cell (RBC) acetylcholinesterase. Wild-type acetylcholinesterase, YT1, has histidine at codon 322, whereas the genetic variant of acetylcholinesterase, YT2, has asparagine. This mutation is located within exon 2 of the ACHE gene, an exon that is present in all alternatively spliced forms of acetylcholinesterase. Therefore, acetylcholinesterase in brain and muscle has the same mutation as RBC acetylcholinesterase. We compared the electrophoretic and kinetic properties of RBC acetylcholinesterases having His 322 or Asn 322. We found no differences in the isoelectric point, mobility on non-denaturing gel electrophoresis, affinity for acetylthiocholine, activity per milligram of RBC ghost protein, substrate inhibition constants, binding to the peripheral site ligand (propidium), and binding to active site ligands (tetrahydroaminoacridine and edrophonium). Thus, although the point mutation elicits antibody production in nonmatching blood transfusion recipients, it has no effect on the enzymatic properties of acetylcholinesterase.
ESTHER : Masson_1994_Blood_83_3003
PubMedSearch : Masson_1994_Blood_83_3003
PubMedID: 8180397

Title : Pressure and propylene carbonate denaturation of native and aged phosphorylated cholinesterase - Masson_1994_J.Mol.Biol_238_466
Author(s) : Masson P , Gouet P , Clery C
Ref : Journal of Molecular Biology , 238 :466 , 1994
Abstract : Organophosphate-inhibited cholinesterases may become progressively refractory to reactivation by nucleophilic compounds due to the dealkylation of an alkoxy group from the covalently bound phosphonate ester. This process is termed "aging". It has been found that "aged" cholinesterases are more resistant to protein unfolding than the non-inhibited ones. The pressure-induced denaturation of the native (non-inhibited) and "aged" tetrameric form of human plasma butyrylcholinesterase was investigated in the presence and absence of a denaturing agent (propylene carbonate). This study was undertaken to determine whether the stability of aged butyrylcholinesterase varies with the structure of the alkyl/aryl (R2) group remaining attached to the phosphorus atom of the organophosphoryl moiety. "Aged" organophosphoryl-cholinesterase conjugates were formed by reacting the enzyme with organophosphates: soman (trimethylpropylmethyl-phosphonofluoridate), sarin (isopropylmethyl-phosphonofluoridate), tabun (ethyl-N-dimethyl-phosphoramidocyanidate), DFP (diisopropyl phosphorofluoridate) and PBPDC (pyrenebutyl-phosphorodichloridate). The dual effects of hydrostatic pressure up to 3.5 kbar and propylene carbonate up to 1.2 M were investigated in 10 mM Tris.HCl (pH 7.0). Non-inhibited and aged enzymes were subjected to pressure/propylene carbonate for 12 hours at 20 degrees C. The perturbing effects of this treatment upon cholinesterase structure were analyzed after pressure release by non-denaturing electrophoresis. Pressure and propylene carbonate induced progressive inactivation of the native enzyme. The loss in activity was correlated with irreversible denaturation of the tetramer and its subsequent aggregation. Similarly, pressure and propylene carbonate induced the formation of irreversibly denatured forms of aged butyrylcholinesterase. These denatured forms are partially unfolded enzyme conformations. The native enzyme was found to be more susceptible to denaturation than aged enzymes, with the exception of the PBPDC-aged enzyme. Methyl phosphono adducts, i.e. soman or sarin-aged conjugates were found to be the most stable aged species. Phenomenological analysis of the pressure/propylene carbonate denaturation maps at half-way of the denaturation process indicated that denaturation is a multistep process. The lowest stability of tabun-aged and DFP-aged conjugates suggested that the size, the orientation and the hydrophobicity of the remaining alkyl/aryl chain (R2) of the organophosphoryl moiety play a role in determining the overall stability of aged enzymes. Molecular modelling of aged adducts shed light on steric constraints exerted by the R2 chain on the salt bridge formed between the negatively charged P-O- of the dealkylated organophosphoryl moiety and protonated His438 N epsilon.
ESTHER : Masson_1994_J.Mol.Biol_238_466
PubMedSearch : Masson_1994_J.Mol.Biol_238_466
PubMedID: 8176737

Title : Recombinant human butyrylcholinesterase G390V, the fluoride-2 variant, expressed in Chinese hamster ovary cells, is a low affinity variant - Masson_1993_J.Biol.Chem_268_14329
Author(s) : Masson P , Adkins S , Gouet P , Lockridge O
Ref : Journal of Biological Chemistry , 268 :14329 , 1993
Abstract : Kinetics of recombinant fluoride-2 variant of human butyrylcholinesterase (Gly390 Val) secreted by Chinese hamster ovary cells were compared to recombinant usual and to usual butyrylcholinesterase purified from human plasma. The usual and fluoride-2 variant were indistinguishable with regard to hydrolysis of benzoylcholine (Km = 5 microM), neutral esters, and at high concentrations of acetylthiocholine, propionylthiocholine, and butyrylthiocholine. However, at low substrate concentrations Km values for acetylthiocholine and succinyldithiocholine were 2-6-fold higher for the fluoride-2 variant. pH rate profiles revealed small differences in pKa that could be attributed to changes in the active site histidine environment. On the other hand, Arrhenius plot analysis of o-nitrophenylbutyrate hydrolysis at pH 7.5 showed no difference in activation energy between fluoride-2 and usual butyrylcholinesterases. Both exhibited an anomalous temperature dependence with a wavelike change in activation energy around 18 degrees C. Affinity of the fluoride-2 variant for sodium fluoride, tacrine, dibucaine, amodiaquin, and succinyldicholine was lower than for usual enzyme. Apparent Ki for succinyldicholine was 125 microM for the fluoride-2 variant and 20 microM for the usual enzyme. Organophosphate inhibition showed equivalent reactivity, indicating that the point mutation altered only the binding properties of the variant. Thus, Km and Ki changes explain the succinyldicholine sensitivity of people carrying the fluoride-2 variant.
ESTHER : Masson_1993_J.Biol.Chem_268_14329
PubMedSearch : Masson_1993_J.Biol.Chem_268_14329
PubMedID: 8314794

Title : [Aging of cholinesterase after inhibition by organophosphates]. - Curtil_1993_Ann.Pharm.Fr_51_63
Author(s) : Curtil C , Masson P
Ref : Ann Pharm Fr , 51 :63 , 1993
Abstract : Progressive inhibition of cholinesterases by organophosphates results from phosphorylation of the active-site serine. Phosphorylated cholinesterases may undergo a dealkylation reaction of the organophosphorus moiety leading to "aged" enzyme, i.e. conversion of the inhibited enzyme into a non-reactivable form. Aging occurs rapidly when the inhibitor is soman, a powerful nerve agent. This reaction promotes formation of a salt bridge between the protonated histidine of the active site catalytic triad and a negatively charged oxygen bound to the phosphorus atom. This reaction is accompanied by enzyme conformational and stability changes. In the research of compounds which retard or prevent the dealkylation reaction of organophosphate-cholinesterase conjugates, some allosteric effectors are relatively efficient by decreasing the velocity of the "aging" process. Knowledge of the three-dimensional structure of non-inhibited, inhibited and aged cholinesterases allows to understand the intimate mechanism of irreversible enzyme inhibition. Modeling of enzyme structure in the presence of effectors is essential to find out new therapeutic means against organophosphate poisoning.
ESTHER : Curtil_1993_Ann.Pharm.Fr_51_63
PubMedSearch : Curtil_1993_Ann.Pharm.Fr_51_63
PubMedID: 8250487

Title : Soman inhibition of butyrylcholinesterase in the presence of substrate: pressure and temperature perturbations - Clery_1992_Biochim.Biophys.Acta_1159_295
Author(s) : Clery C , Masson P , Heiber-Langer I , Balny C
Ref : Biochimica & Biophysica Acta , 1159 :295 , 1992
Abstract : Irreversible inhibition of butyrylcholinesterase by soman was studied in the presence of the substrate (o-nitrophenyl butyrate). Inhibition was found of the competitive complexing type. Study at different temperatures and pressures showed that the behavior of the enzyme differs from that of the inhibitor-free enzyme. In the absence of inhibitor, enzyme kinetics displayed a non-linear temperature dependence with a break at 21 degrees C. In the presence of a non-inhibitor structural analog of soman (pinacolyl dimethylphosphinate and methyl dimethylphosphinate), the Arrhenius plot break is slightly shifted (18 degrees C). On the other hand, in the presence of soman this break is abolished. The pressure-dependence of the substrate hydrolysis revealed also differences between the native enzyme and the enzyme in the presence of soman: the sign and magnitude of the apparent activation volume (delta V not equal to) were different for the two reactions. Beyond 300 bar, in the presence of soman, a plateau (delta V not equal to approx. 0) was observed over a large pressure range depending on temperature. Such a behavior with respect to temperature and pressure can reflect a soman-induced enzyme conformational state. Thus, temperature and pressure perturbations of the kinetics allow to complete the inhibition scheme of butyrylcholinesterase by soman. Our data suggest that upon soman binding, the enzyme undergoes a long-lived soman-induced-fit conformational change preceding the phosphonylation step. However, an alternative hypothesis according to which the enzyme processes a secondary soman-binding site cannot be ruled out.
ESTHER : Clery_1992_Biochim.Biophys.Acta_1159_295
PubMedSearch : Clery_1992_Biochim.Biophys.Acta_1159_295
PubMedID: 1390935

Title : Expression and Refolding of Functional Human Butyrylcholinesterase from E. Coli -
Author(s) : Masson P , Adkins S
Ref : In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases , (Shafferman, A. and Velan, B., Eds) Plenum Press, New York :49 , 1992
PubMedID:

Title : Molecular Heterogeneity of Human Plasma Cholinesterase -
Author(s) : Masson P
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :42 , 1991
PubMedID:

Title : Poster: Stability of native and organophosphate-inhibited butyry1cholinesterase under high pressure -
Author(s) : Masson P , Clery C , Huchet D
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :275 , 1991
PubMedID:

Title : A comparative Raman spectroscopic study of cholinesterases - Aslanian_1991_Biochimie_73_1375
Author(s) : Aslanian D , Grof P , Bon S , Masson P , Negrerie M , Chatel JM , Balkanski M , Taylor P , Massoulie J
Ref : Biochimie , 73 :1375 , 1991
Abstract : We report Raman spectra of various cholinesterases: lytic tetrameric forms (G4) obtained by tryptic digestion of asymmetric acetylcholinesterase (AChE) from Torpedo californica and Electrophorus electricus, a PI-PLC-treated dimeric form (G2) of AChE from T marmorata, and the soluble tetrameric form (G4) of butyrylcholinesterase (BCHE) from human plasma. The contribution of different types of secondary structure was estimated by analyzing the amide I band, using the method of Williams. The spectra of cholinesterases in 10 mM Tris-HCl (pH 7.0) indicate the presence of both alpha-helices (about 50%) and beta-sheets (about 25%), together with 15% turns and 10% undefined structures. In 20 mM phosphate buffer (pH 7.0), the spectra indicated a smaller contribution of alpha-helical structure (about 35%) and an increased beta-sheet content (from 25 to 35%). This shows that the ionic milieu profoundly affects either the conformation of the protein (AChE activity is known to be sensitive to ionic strength), or the evaluation of secondary structure, or both. In addition, we analyzed vibrations corresponding to the side chains of aromatic and aliphatic amino acids. In particular, the analyses of the tyrosine doublet (830-850 cm-1) and of the tryptophan vibration at 880 cm-1 indicated that these residues are predominantly 'exposed' on the surface of the molecules.
ESTHER : Aslanian_1991_Biochimie_73_1375
PubMedSearch : Aslanian_1991_Biochimie_73_1375
PubMedID: 1799630

Title : Poster: Use of the polymerase chain reaction for homology probing of butyrylchoIinesterase (BCHE) in several animal species -
Author(s) : Arpagaus M , Vaughan TA , La Du BN , Lockridge O , Masson P , Chatonnet A , Newton M , Taylor P
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :194 , 1991
PubMedID:

Title : Structural and functional investigations of cholinesterases by means of affinity electrophoresis - Masson_1991_Cell.Mol.Neurobiol_11_173
Author(s) : Masson P
Ref : Cellular Molecular Neurobiology , 11 :173 , 1991
Abstract : 1. After a brief survey of the basic affinity electrophoresis concepts, the usual ways for preparing affinity electrophoresis ligands are examined. 2. Then results obtained on cholinesterases are reviewed. This section includes (a) structural and functional investigations on anionic sites, i.e., study of ligand-induced conformational change, organophosphate-induced "aging," genetic variants, and active-site topology; and (b) characterization of cholinesterase conjugates (hybrid proteins) and glycoinositol phospholipid-anchored cholinesterases. 3. The future prospects of affinity electrophoresis, e.g., investigations on the esteratic site and exploration of the carbohydrate moiety, are emphasized in the concluding section.
ESTHER : Masson_1991_Cell.Mol.Neurobiol_11_173
PubMedSearch : Masson_1991_Cell.Mol.Neurobiol_11_173
PubMedID: 1849453

Title : Poster: Molecular characterization of the C5 human plasma cholinesterase variant -
Author(s) : Masson P , Froment MT , Audras JC , Renault F
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :60 , 1991
PubMedID:

Title : Poster: A comparative raman spectroscopic study of cholinesterases -
Author(s) : Aslanian D , Grof P , Masson P , Taylor P , Bon S , Chatel JM , Massoulie J
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :56 , 1991
PubMedID:

Title : Use of the polymerase chain reaction for homology probing of butyrylcholinesterase from several vertebrates - Arpagaus_1991_J.Biol.Chem_266_6966
Author(s) : Arpagaus M , Chatonnet A , Masson P , Newton M , Vaughan TA , Bartels CF , Nogueira CP , La Du BN , Lockridge O
Ref : Journal of Biological Chemistry , 266 :6966 , 1991
Abstract : Genomic blots from man, monkey, cow, sheep, pig, rabbit, dog, rat, mouse, guinea pig, and chicken DNA were hybridized with probes derived from the four exons of the human butyrylcholinesterase gene (BCHE) (Arpagaus, M., Kott, M., Vatsis, K. P., Bartels, C. F., La Du, B. N., and Lockridge, O. (1990) Biochemistry 29, 124-131). Results showed that the BCHE gene was present in a single copy in the genome of all these vertebrates. The polymerase chain reaction was used to amplify genomic DNA from these animals with oligonucleotides derived from the human BCHE coding sequence. The amplified segment contained 423 bp of BCHE sequence including the active site serine of the enzyme (amino acid 198) and a component of the anionic site, aspartate 70. Amplification was successful for monkey, pig, cow, dog, sheep, and rabbit DNA, but unsuccessful for rat, guinea pig, mouse, and chicken DNA. Amplified segments were cloned in M13 and sequenced. The mouse sequence was obtained by sequencing a genomic clone. The highest identity of the human amino acid sequence was found with monkey (100%) and the lowest with mouse (91.5%). The sequence around the active site serine 198, Phe-Gly-Glu-Ser-Ala-Gly-Ala, was conserved in all eight animals as was the anionic site component, aspartate 70. A phylogenetic tree of mammalian butyrylcholinesterases was constructed using the partial BCHE sequences.
ESTHER : Arpagaus_1991_J.Biol.Chem_266_6966
PubMedSearch : Arpagaus_1991_J.Biol.Chem_266_6966
PubMedID: 2016308
Gene_locus related to this paper: bovin-BCHE , canfa-BCHE , macmu-BCHE , pig-BCHE , sheep-BCHE

Title : Evidence for a single butyrylcholinesterase gene in individuals carrying the C5 plasma cholinesterase variant (CHE2) - Masson_1990_FEBS.Lett_262_115
Author(s) : Masson P , Chatonnet A , Lockridge O
Ref : FEBS Letters , 262 :115 , 1990
Abstract : DNA of 3 unrelated individuals carrying the human plasma butyrylcholinesterase C5 variant (CHE2) was isolated from white blood cells. Southern blot patterns of DNA restriction fragments probed with each of the 4 butyrylcholinesterase exons provided evidence that the production of C5 is not directed by a second butyrylcholinesterase gene. This finding supports the suggestion that the C5 variant is a hybrid enzyme resulting from the association of butyrylcholinesterase subunits with a non-cholinesterase protein.
ESTHER : Masson_1990_FEBS.Lett_262_115
PubMedSearch : Masson_1990_FEBS.Lett_262_115
PubMedID: 2318303

Title : Monoclonal antibodies allow precipitation of esterasic but not peptidasic activities associated with butyrylcholinesterase - Checler_1990_J.Neurochem_55_750
Author(s) : Checler F , Grassi J , Masson P , Vincent JP
Ref : Journal of Neurochemistry , 55 :750 , 1990
Abstract : Commercially available and affinity-purified butyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine-enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucine-enkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90- and 180-kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG-purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide-hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities of this enzyme.
ESTHER : Checler_1990_J.Neurochem_55_750
PubMedSearch : Checler_1990_J.Neurochem_55_750
PubMedID: 1696618

Title : Conformational plasticity of butyrylcholinesterase as revealed by high pressure experiments - Masson_1990_Biochim.Biophys.Acta_1041_223
Author(s) : Masson P , Balny C
Ref : Biochimica & Biophysica Acta , 1041 :223 , 1990
Abstract : The ligand binding and kinetic behaviour of butyrylcholinesterase (EC 3.1.1.8, acylcholine acylhydrolase) from human plasma was studied at 35 degrees C under high hydrostatic pressure. The binding of phenyltrimethylammonium was studied by affinity electrophoresis at various pressures ranging from 10(-3) to 2 kbar. The kinetics of enzyme carbamylation with N-methyl(7-dimethylcarbamoxy)quinolinium iodide was studied in single-turnover conditions up to 1.2 kbar using a high-pressure stopped-flow fluorimeter. Experiments were carried out in different media: 1 mM Tris-HCl (pH 8) with water, water containing 0.1 M lithium chloride and deuterium oxide as solvents. The volume changes (delta V and delta V++) associated with each process were determined from the pressure-dependence of the binding and kinetic constants. Kinetic data show that the binding of substrate to the enzyme leads to a pressure-sensitive enzyme conformational state which cannot accomplish the catalytic act. The pressure-induced inhibitory effect is highly cooperative; it depends on both the nature (charged or neutral) and the concentration of the substrate. Also, large solvent effects indicate that enzyme sensitivity to pressure depends on the solvent structure. This findings suggests that the substrate-dependent pressure effect is modulated by the solvation state of the enzyme.
ESTHER : Masson_1990_Biochim.Biophys.Acta_1041_223
PubMedSearch : Masson_1990_Biochim.Biophys.Acta_1041_223
PubMedID: 2268667

Title : A naturally occurring molecular form of human plasma cholinesterase is an albumin conjugate - Masson_1989_Biochim.Biophys.Acta_998_258
Author(s) : Masson P
Ref : Biochimica & Biophysica Acta , 998 :258 , 1989
Abstract : Human plasma cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) consists of four main molecular forms designated as C1, C2, C3 and C4 according to their electrophoretic mobility on gels. The major component, C4, is the tetrameric form; C1 and C3 are the monomeric and dimeric forms, respectively. The C2 form, which has an apparent free electrophoretic mobility higher than that of the three size isomers, and, moreover, a higher isoelectric point, was found to be a covalent conjugate between the cholinesterase monomer and serum albumin. This result is supported by the following arguments: the non-catalytic subunit of C2 was found to be a carbohydrate-free protein of apparent molecular mass 65 kDa that could not be labelled by diisopropylfluorophosphonate in the labelling conditions of esterases. It possesses a high affinity for a long-chain aliphatic ligand (a substituted octadecylamine) and for Cibacron blue F3 GA, and could be adsorbed on an immunoadsorbent for albumin. The two subunits of C2 are disulfide bridge linked; the active center of the cholinesterase subunit is partly masked by the albumin molecule. The conjugation reaction very likely occurs in the hepatic cell and not in plasma.
ESTHER : Masson_1989_Biochim.Biophys.Acta_998_258
PubMedSearch : Masson_1989_Biochim.Biophys.Acta_998_258
PubMedID: 2553123

Title : Stability of butyrylcholinesterase: thermal inactivation in water and deuterium oxide - Masson_1988_Biochim.Biophys.Acta_957_111
Author(s) : Masson P , Laurentie M
Ref : Biochimica & Biophysica Acta , 957 :111 , 1988
Abstract : Irreversible thermal inactivation of the tetrameric form of human plasma butyrylcholinesterase (cholinesterase; EC 3.1.1.8) was studied in water and in deuterium oxide at pH 7 in the temperature range 53-65 degrees C. The enzyme inactivation follows a complex kinetics that may be described by the sum of two apparent first-order processes. The Eyring plot for enzyme inactivation exhibits a wavelike discontinuity over a span of 2 C degrees around 58 degrees C. This transition was interpreted in terms of equilibrium between two temperature-dependent conformational states. Though 2H2O does not alter the overall multistep inactivation process, a slight solvent isotope effect was observed: a stabilizing effect and a shift in the transition temperature. A comparison between several enzyme preparations revealed differences in thermodynamic activation parameters of inactivation suggesting microheterogeneity in enzyme structures. Kinetics of inactivation of usual (E1uE1u) and atypical (E1aEa1a++) enzymes were compared. The atypical enzyme was found to be more stable than the usual phenotype.
ESTHER : Masson_1988_Biochim.Biophys.Acta_957_111
PubMedSearch : Masson_1988_Biochim.Biophys.Acta_957_111
PubMedID: 3179317

Title : Effects of high pressure on the single-turnover kinetics of the carbamylation of cholinesterase - Masson_1988_Biochim.Biophys.Acta_954_208
Author(s) : Masson P , Balny C
Ref : Biochimica & Biophysica Acta , 954 :208 , 1988
Abstract : Pressure, as a perturbing variable, is one of the most powerful tools to investigate the thermodynamic parameters of chemical reactions and to study the mechanism of enzyme-catalyzed reactions. The effect of elevated hydrostatic pressure (up to 0.8 kbar) on the reaction of butyrylcholinesterase with N-methyl-(7-dimethylcarbamoxy)quinolinium was determined under single-turnover conditions at 35 degrees C. The rate of carbamylation was monitored as the accumulation of the fluorescent ion, N-methyl-7-hydroxyquinolinium, in a high-pressure stopped-flow apparatus designed for the assay of fluorescence. Elevated pressure favored formation of the enzyme-substrate complex but inhibited carbamylation of the enzyme. Because a single reaction step was recorded, it was possible to interpret the data obtained under high pressure in the form of Michaelis-Menten equations. From the pressure dependence of the dissociation constant for the enzyme-substrate complex and the rate constant for carbamylation, maximal volume changes accompanying these events were determined. The value for the binding process, delta Vb = -129 ml.mol-1, is too large to be related only to volumetric changes in the active center. Substrate-induced conformational change and change of water structure appear to be the dominant contributions to the overall volume change associated with substrate binding. The large positive activation volume measured (delta V not equal to = 119 ml.mol-1) may also reflect extended structural and hydration changes. At pressures greater than 0.4 kbar, an additional pressure effect, dependent on substrate concentration, occurred in a narrow pressure interval. This effect may have resulted from a substrate-induced pressure-sensitive enzyme conformational state.
ESTHER : Masson_1988_Biochim.Biophys.Acta_954_208
PubMedSearch : Masson_1988_Biochim.Biophys.Acta_954_208
PubMedID: 3365437

Title : Kinetic evidence for thermally induced conformational change of butyrylcholinesterase - Ferro_1987_Biochim.Biophys.Acta_916_193
Author(s) : Ferro A , Masson P
Ref : Biochimica & Biophysica Acta , 916 :193 , 1987
Abstract : The effect of temperature on the kinetics of human plasma butyrylcholinesterase-catalyzed reactions was studied. The Arrhenius plot of o-nitrophenylbutyrate hydrolysis presents a break at 21 degrees C. However, nucleophilic competition data indicate that there is no change in the rate-limiting step of the overall reaction. In addition, the temperature dependence of the bimolecular rate constant of enzyme carbamylation shows a break at 18 degrees C. These results argue for the existence of thermally induced conformational active states of the enzyme tetramer. It is suggested that the effects of this transition on kinetics arise at the acylation step.
ESTHER : Ferro_1987_Biochim.Biophys.Acta_916_193
PubMedSearch : Ferro_1987_Biochim.Biophys.Acta_916_193
PubMedID: 3676330

Title : Evidence that the conformational stability of 'aged' organophosphate-inhibited cholinesterase is altered - Masson_1986_Biochim.Biophys.Acta_869_304
Author(s) : Masson P , Goasdoue JL
Ref : Biochimica & Biophysica Acta , 869 :304 , 1986
Abstract : In order to determine whether a structural modification at the active center of cholinesterase may alter the conformational stability of the enzyme we compared the urea-induced unfolding of the tetrameric form of non-inhibited and irreversibly inhibited human plasma cholinesterase (acylcholine acylhydrolase, EC 3.1.1.8). We studied enzyme inhibited by methanesulfonyl fluoride, diisopropylfluorophosphonate (DFP) and racemic soman. DFP- and soman-inhibited cholinesterases are converted spontaneously into non-reactivable forms called 'aged' enzymes through a process involving dealkylation of the bound organophosphate residue. The unfolding was followed by transverse urea-gradient polyacrylamide electrophoresis at various temperatures ranging from 0 to 60 degrees C. Unfolding of cholinesterase appears to be a complex process. The denaturation patterns showed that partially unfolded states are thermodynamically unstable, but that several intermediates are involved; the lifetime of these depends on the temperature at which electrophoreses are carried out. Cholinesterase inhibited by methanesulfonyl fluoride behaved like the non-inhibited enzyme. On the other hand, small but significant differences in stability between non-inhibited and aged enzymes were observed. Whatever the temperature, the urea concentration at the mid-point of transition was always greater for aged enzyme than for the non-inhibited enzyme. In addition, aged enzymes showed more complex denaturation patterns at the lower temperatures (under 20 degrees C). These findings suggest that the overall stability of aged-cholinesterases is slightly increased as compared with the stability of non-inhibited or methanesulfonyl fluoride-inhibited enzymes. The denaturation pattern obtained at 0 degree C for soman-inhibited cholinesterase under non-aging conditions (inhibition at 0 degree C, pH 10.7) was similar to that of non-inhibited enzyme at this temperature, although splitting in two of the denaturation curve over the transition zone reflects the heterogeneity of soman-inhibited enzyme. The slight difference in denaturation behavior between these species may be due to stereoisomerism in soman. The differences in electrophoretic behavior and apparent stability observed between non-inhibited and aged enzymes were interpreted as the result of a conformational change induced by the dealkylation reaction of enzyme-inhibitor conjugates.
ESTHER : Masson_1986_Biochim.Biophys.Acta_869_304
PubMedSearch : Masson_1986_Biochim.Biophys.Acta_869_304
PubMedID: 3947640

Title : Is the peptidase activity of highly purified human plasma cholinesterase due to a specific cholinesterase isoenzyme or a contaminating dipeptidylaminopeptidase? - Chatonnet_1986_Biochimie_68_657
Author(s) : Chatonnet A , Masson P
Ref : Biochimie , 68 :657 , 1986
Abstract : The peptidase site of human plasma cholinesterase (butyrylcholinesterase) is distinct from its esteratic site. We found that the number of peptidase sites on an enzyme highly purified from pooled plasma is less than 0.1, as compared with 4 esteratic sites, per tetramer. However, the subunits which carry the peptidase sites are electrophoretically indistinguishable from esteratic subunits. The atypical-silent enzyme (Ea1Es1) had a much higher absolute peptidase activity when substance P was used as the substrate, and we found that the number of peptidase and esteratic sites of this enzyme was roughly the same. This suggests that the mutated esteratic site of the silent possesses a peptidase activity. The esteratic site of the usual allozyme (Eu1Eu1) has no peptidase activity towards substance P. However, a small proportion of peptidase subunits are present in all preparations of enzymes purified from the plasmas of homozygote individuals. The peptidase activity of butyrylcholinesterase might therefore correspond to a specific isoenzyme produced by an epigenetic mechanism or produced by a gene distinct from genes E1 and E2 encoding for cholinesterase subunits. However, the possibility that highly purified cholinesterase contains traces of a dipeptidylaminopeptidase cannot be completely ruled out.
ESTHER : Chatonnet_1986_Biochimie_68_657
PubMedSearch : Chatonnet_1986_Biochimie_68_657
PubMedID: 2425854

Title : Thermodynamic arguments for temperature-induced cryptic conformational change of human plasma cholinesterase - Masson_1986_Biochim.Biophys.Acta_874_90
Author(s) : Masson P , Balny C
Ref : Biochimica & Biophysica Acta , 874 :90 , 1986
Abstract : The temperature and pressure dependence of the kinetics of the hydrolysis of o-nitrophenylbutyrate by human plasma tetrameric form cholinesterase (EC 3.1.1.8) was studied. The study was carried out on the one hand at atmospheric pressure by spectrophotometry at various temperatures ranging from 0 to 40 degrees C and, on the other hand by high-pressure stopped-flow spectrophotometry at 3.5, 25 and 35 degrees C in the pressure range 10(-3) to 2 kbar. The Arrhenius plot showed a break at 21 +/- 1 degrees C. Kinetic parameters, activation parameters and volume changes are reported. Discontinuities in the thermodynamic quantities obtained from temperature and pressure (up to 0.8 kbar) dependence of hydrolysis rates are discussed; they have been interpreted as the result of a temperature-induced cryptic conformational change of the enzyme at around 20 degrees C. Beyond 1 kbar the kinetics exhibited several complexities: curvature of the progress curves and high positive or negative activation volume changes depending on temperature and substrate concentration. These complex interacting effects between temperature, pressure and substrate concentration are discussed.
ESTHER : Masson_1986_Biochim.Biophys.Acta_874_90
PubMedSearch : Masson_1986_Biochim.Biophys.Acta_874_90
PubMedID: 3768380

Title : [Affinity electrophoresis in polyacrylamide gel. Influence of the concentration in the gel on the apparent affinity of cholinesterase for an anionic ligand site]. [French] - Masson_1985_J.Chromato_328_135
Author(s) : Masson P , Marnot B
Ref : Journal of Chromatography , 328 :135 , 1985
Abstract : Affinity electrophoresis of three purified molecular forms of human plasma cholinesterase (monomer C1, dimer C3, tetramer C4) was carried out in polyacrylamide gels at various total acrylamide concentrations ranging from 3.48 to 9% in a discontinuous buffer system. A water-soluble linear copolymer supporting procainamide, a ligand of the anionic site of cholinesterase, was physically entrapped at various concentrations within the gel network. The combined effects of gel concentration and ligand concentration on the affinity pattern of the three molecular forms were studied. It was found that gel concentration influences the apparent binding activity of their anionic site: The apparent strength of interaction varied with the gel concentration: the denser the gel was, the higher the apparent affinity. The ligand-induced isomerization process was also depending on the gel concentration: the ligand concentration from which each zone is splitting into two moving zones decreased as the total gel concentration increased. These results show that the electrophoretic matrix plays an important role in the affinity process in affinity electrophoresis presumably by controlling kinetic effects: kinetics of protein-ligand complex formation and dissociation reactions, and mass transfer kinetics.
ESTHER : Masson_1985_J.Chromato_328_135
PubMedSearch : Masson_1985_J.Chromato_328_135
PubMedID: 4030968

Title : Study of the peptidasic site of cholinesterase: preliminary results - Chatonnet_1985_FEBS.Lett_182_493
Author(s) : Chatonnet A , Masson P
Ref : FEBS Letters , 182 :493 , 1985
Abstract : The peptidasic site of highly purified human plasma cholinesterase was investigated using active-site-directed inhibitors. Peptidase activity was assayed taking substance P as substrate. Inhibition by organophosphates indicated that the peptidasic site contained an active serine. The presence of essential histidine residues associated with serine was revealed by histidine modifications. Carboxyl group reagents showed that the active centre contained carboxyl groups in a non-polar environment. The removal of sialic acids did not alter peptidase activity. The peptidasic site of cholinesterase shared many properties with serine proteases sites and esteratic sites of cholinesterases. In addition, with the peptidasic site, as well as the esteratic site, there was always the possibility of 'aging' when inhibited by DFP or soman.
ESTHER : Chatonnet_1985_FEBS.Lett_182_493
PubMedSearch : Chatonnet_1985_FEBS.Lett_182_493
PubMedID: 2579854

Title : [The peptidase site of butyrylcholinesterase is distinct from the esterase site]. [French] - Chatonnet_1984_C.R.Acad.Sci.III_299_529
Author(s) : Chatonnet A , Masson P
Ref : Comptes Rendus de l Academie des Sciences , 299 :529 , 1984
Abstract : Highly purified human plasma butyrylcholinesterase was inhibited by reversible inhibitors of esterase activity and modified by active-site-directed irreversible inhibitors of esterases and proteases. Peptidase and esterase activities of inhibited enzyme were simultaneously essayed from rates of hydrolysis of substance P (first cleavage) and butyrylthiocholine respectively. Inhibition parameters values and rates of inactivation of the two activities provide evidence that the peptidasic site is distinct from the esteratic site.
ESTHER : Chatonnet_1984_C.R.Acad.Sci.III_299_529
PubMedSearch : Chatonnet_1984_C.R.Acad.Sci.III_299_529
PubMedID: 6208983

Title : [Electrophoretic study of aged butyrylcholinesterase after inhibition by soman]. [French] - Masson_1984_Biochimie_66_235
Author(s) : Masson P , Marnot B , Lombard JY , Morelis P
Ref : Biochimie , 66 :235 , 1984
Abstract : The following states of purified tetrameric form (C4) of human plasma butyrylcholinesterase were studied by electrophoretic techniques: native, inhibited by soman and by methane sulfonyl fluoride and soman-aged. In order to detect a significant conformational change of the aged cholinesterase as compared to the non-inhibited (native) species, enzymes were treated with a set of bifunctional reagents (diimidates) of different chain lengths. After denaturation, the cross-link products were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The peak areas of the cross-linked species and the parameters of cross-linkability were calculated from densitometric data, versus the maximal effective reagent length. The effect of occupancy of the esteratic site by substituted phosphonyl group and by methyl-sulfonyl residue on the binding activity of the anionic site was studied by affinity electrophoresis at varying temperatures with immobilized-procanamide as ligand. Apparent dissociation-constants of the enzyme-ligand complexes were estimated from measurement of mobilities versus ligand concentration. Corresponding thermodynamic quantities were calculated from Van't Hoff plots and basic thermodynamic equations. The reactivity of aged-cholinesterase with diimidates was similar to that of the native enzyme. Affinity for immobilized-procanamide was slightly lowered in aged and inhibited enzymes as compared to the native and sulfonylated enzymes. As for the ligand-induced isomerization of anionic site (A----B), revealed by affinity electrophoresis, the ligand concentration at the midpoint of transition (A = 0,5) was slightly greater for the aged enzyme than for the native one. From these results, the following conclusions can be drawn: the dealkylation of soman-cholinesterase conjugate (aging) does not seem to induce structural changes detectable in the cross-linkability of lysyle residues at the subunit interfaces and on the surface of the tetrameric enzyme. On the other hand, the affinity of the anionic site and ligand-induced isomerization process are altered in soman-inhibited and aged enzymes. These data suggest the occurrence of a weak conformational change of the active center and/or the formation of non-covalent bonds between the methylphosphonyl residue and side chain groups as a result of the dealkylation reaction.
ESTHER : Masson_1984_Biochimie_66_235
PubMedSearch : Masson_1984_Biochimie_66_235
PubMedID: 6331528

Title : Poster 84. Electrophoretic studies of the aged soman-inhibited butyrylcholinesterase -
Author(s) : Masson P , Marnot B , Lombard JY , Morelis P
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter , 1984
PubMedID:

Title : [Possibilities of study of the human plasma cholinesterase variants by affinity electrophoresis]. [French] - Masson_1983_J.Chromato_273_289
Author(s) : Masson P , Vallin P
Ref : Journal of Chromatography , 273 :289 , 1983
Abstract : Affinity electrophoresis has been applied to the study of the multiple molecular forms of three human plasma cholinesterase phenotypes (usual enzyme U, atypical enzyme A and intermediate UA). Electrophoreses were carried out in polyacrylamide gels containing a water-soluble macromolecular derivative of m-amino-(substituted)-phenyltrimethylammonium immobilized within the gel network. Apparent dissociation constants (KD app) were estimated from the mobilities of the enzymes versus ligand concentration. The ratio of KD app values of the molecular forms of phenotypes A and U which is approximately 2 is consistent with the hypothesis that the anionic site is altered in atypical enzyme.
ESTHER : Masson_1983_J.Chromato_273_289
PubMedSearch : Masson_1983_J.Chromato_273_289
PubMedID: 6863445

Title : [Multiple molecular forms of human plasma butyrylcholinesterase. II.-Study of the C1, C3 and C4 components by means of affinity electrophoresis (author's transl)]. [French] - Masson_1982_Biochim.Biophys.Acta_701_269
Author(s) : Masson P , Privat de Garilhe A , Burnat P
Ref : Biochimica & Biophysica Acta , 701 :269 , 1982
Abstract : Affinity electrophoresis has been applied to the analysis of the multiple molecular forms of human plasma cholinesterase allozyme U. A water-soluble p-amino-substituted-phenyltrimethylammonium polyacrylamide was synthetized by copolymerization of an unsaturated derivative of the ligand with acrylamide, and entrapped at various concentrations within the matrix of separating gels. Electrophoresis was carried out in these gels, and the relative mobility of the molecular forms of the enzyme was decreased. From the variation of mobility (Rm) as a function of immobilized ligand concentrations, the apparent dissociation constants of monomer (C1), dimer (C3) and tetramer (C4) of phenotype U were calculated. The decrease in mobility was reversed by addition of non-immobilized competitive ligands (N-methylpyridinium and N-methylacridinium). The appearance of the slopes of Rmi-1 vs. concentration does not give sufficient information for determination of the number of anionic binding sites of C4, but the slight curvature of the plots suggests that bivalent or higher interactions occur when the concentration is sufficiently high. For all three size isomers from a critical ligand concentration, a second zone, named B, appears and intensifies rapidly at the expense of the first zone (A) as the immobilized ligand concentration increases. Among several possible explanations of this phenomenon, it is proposed that the ligand induces a conformational isomerization of the enzymes with a change in affinity (KD,B less than KD,A) and that the interconversion process between the two states B in equilibrium A is slow compared with the ligand-association equilibrium dissociation steps.
ESTHER : Masson_1982_Biochim.Biophys.Acta_701_269
PubMedSearch : Masson_1982_Biochim.Biophys.Acta_701_269
PubMedID: 7066331

Title : [Purification of butyrylcholinesterase from human plasma]. [French] - Masson_1980_C.R.Acad.Sci.III_290_857
Author(s) : Masson P , Sussmilch A , Charlet JP
Ref : Comptes Rendus de l Academie des Sciences , 290 :857 , 1980
Abstract : Human plasma butyrylcholinesterase (E.C.3.1.1.8) was highly purified by a three-step procedure involving affinity and ion-exchange chromatography techniques. The final product was about 15,000-fold purified with a yield of 12% and a specific activity of 450 U/mg.
ESTHER : Masson_1980_C.R.Acad.Sci.III_290_857
PubMedSearch : Masson_1980_C.R.Acad.Sci.III_290_857
PubMedID: 6771034

Title : [Multiple molecular forms of human plasma butyrylcholinesterase. I. Apparent molecular parameters and broad pattern of the quaternary structure (author's transl)]. [French] - Masson_1979_Biochim.Biophys.Acta_578_493
Author(s) : Masson P
Ref : Biochimica & Biophysica Acta , 578 :493 , 1979
Abstract : Apparent molecular parameters (molecular weights, sedimentation constants, partial specific volumes, free electrophoretic mobilities and isoelectric points) of the four molecular forms C-1, C-2, C-3 and C-4 of human plasma butyrylcholinesterase (EC 3.1.1.8) have been demonstrated by polyacrylamide gel electrophoresis methods and centrifugation in sucrose gradient. The C-1 component is the monomeric form of the enzyme )Mr = 84 800 +/- 5800). All the forms are partially interconvertible and C-1, C-3, C-4 are size isomers corresponding to the monomer, dimer and tetramer of the enzyme. An estimation of the general shape of these forms attempted from electrophoretic and hydrodynamic parameters suggests that they are prolate ellipsoids. The C-4 component in which the axial ratio is at least equal to 8 appears to be arranged as a dimer of dimers (C-3)2 in which the two units are associated in a quasi-linear fashion. The C-2 component is composed of C-1 associated with an inactive smaller subunit, which is responsible for its specific electrical properties (mobility and isoelectric point).
ESTHER : Masson_1979_Biochim.Biophys.Acta_578_493
PubMedSearch : Masson_1979_Biochim.Biophys.Acta_578_493
PubMedID: 486534

Title : [Determination of free electrophoretic mobility of proteins by gradient polyacrylamide electrophoresis]. [French] -
Author(s) : Masson P
Ref : Journal of Chromatography , 169 :386 , 1979
PubMedID: 536428

Title : [Frequency of locus E1 variants of plasma butyrylcholinesterase in a French population]. [French] - Masson_1979_C.R.Acad.Sci.III_289_537
Author(s) : Masson P , Germani F , Plasse M
Ref : Comptes Rendus de l Academie des Sciences , 289 :537 , 1979
Abstract : Human plasma cholinesterase (E.C.3.1.1.8) from 1,594 blood donors was phenotyped on the basis of dibucane, fluoride, chloride and succinylcholine differential inhibitions according to the criteria of Brown et coll. The observed gene frequencies are: E1u = 0.970,8, E1a = 0.188,0, E1f = 0.103,0.
ESTHER : Masson_1979_C.R.Acad.Sci.III_289_537
PubMedSearch : Masson_1979_C.R.Acad.Sci.III_289_537
PubMedID: 118816