Ordentlich A

General

Full name : Ordentlich Arie

First name : Arie

Mail : Israel Institute for Biological Research, P.O. Box 19, Ness-Ziona 70450, Israel

Zip Code :

City :

Country : Israel

Email : ordentl@.sharmin

Phone : 972-8-381442

Fax : 972-8-401404

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References (70)

Title : Next generation OP-bioscavengers: a circulatory long-lived 4-PEG hypolysine mutant of F338A-HuAChE with optimal pharmacokinetics and pseudo-catalytic characteristics - Kronman_2010_Chem.Biol.Interact_187_253
Author(s) : Kronman C , Cohen O , Mazor O , Ordentlich A , Raveh L , Velan B , Shafferman A
Ref : Chemico-Biological Interactions , 187 :253 , 2010
Abstract : We have shown previously that conjugation of polyethylene glycol (PEG) chains to recombinant human acetylcholinesterase (rHuAChE) results in the extension of its residence time in the circulation of mice and monkeys [1,2]. By profiling the pharmacokinetic behavior of an array of well-defined hypolysine human mutant AChE molecules following PEGylation, we now determine that the duration of these enzyme forms in the circulation of rhesus macaques correlates with their number of appended PEG moieties, and is influenced by the actual location of the PEG chains at the molecule surface, as well. These findings, which concur with those we have previously established in mice, indicate that a common set of rules dictates the circulatory fate of PEGylated HuAChEs in rodents and non-human primates. In addition to its effect on circulatory residence, PEGylation reduces the ability of the rHuAChE bioscavenger to elicit an immune response in the heterologous mouse animal system. Thus, an inverse relationship between anti-AChE antibody production and PEG loading was observed following repeated administration of the different PEGylated hypolysine human AChEs to mice. We note however, that in rhesus macaques, the essentially homologous (human) AChE does not induce specific anti-AChE antibodies after repeated administration of high doses of the enzyme in its PEGylated form, and even in its non-PEGylated form. Taken together, these findings indicate that PEG acts by veiling enzyme-related epitopes, which would otherwise interact with host circulatory elimination pathways and immune system. The barring of such interactions by obstructive PEGs, confers the enzyme molecule with both extended circulatory residence and mitigated immunogenic properties. Further modulation by incorporation of the F338A mutation into the PEGylated hypolysine rHuAChE enzyme mold, resulted in the generation of an OP-bioscavenger that displayed reduced aging rates and could effectively protect mice against repeated exposure to CW agents. This selected 4-PEG F338A-AChE can serve as a paradigm for new generation OP-bioscavengers, specifically tailored for prophylactic treatment against toxic OP-agents.
ESTHER : Kronman_2010_Chem.Biol.Interact_187_253
PubMedSearch : Kronman_2010_Chem.Biol.Interact_187_253
PubMedID: 20005217

Title : Effective post-exposure protection against lethal orthopoxviruses infection by vaccinia immune globulin involves induction of adaptive immune response - Lustig_2009_Vaccine_27_1691
Author(s) : Lustig S , Maik-Rachline G , Paran N , Melamed S , Israely T , Erez N , Orr N , Reuveny S , Ordentlich A , Laub O , Shafferman A , Velan B
Ref : Vaccine , 27 :1691 , 2009
Abstract : The therapeutic potential of human vaccinia immunoglobulin (VIG) in orthopoxvirus infection was examined using two mouse models for human poxvirus, based on Ectromelia virus and Vaccinia Western Reserve (WR) respiratory infections. Despite the relatively fast clearance of human VIG from mice circulation, a single VIG injection protected immune-competent mice against both infections. Full protection against lethal Ectromelia virus infection was achieved by VIG injection up to one day post-exposure, and even injection of VIG two or three days post-infection conferred solid protection (60-80%). Nevertheless, VIG failed to protect VACV-WR challenged immune-deficient mice, even though repeated injections prolonged SCID mice survival. These results suggest the involvement of host immunity in protection. VIG provides the initial protective time-window allowing induction of the adaptive response required to achieve complete protection. Additionally, VIG can be administered in conjunction with active Vaccinia-Lister vaccination. Vaccine efficiency is not impaired, providing a non-prohibitive VIG dose is used. Thus, VIG can be used as a prophylactic measure against post-vaccinal complications but could also serve for post-exposure treatment against smallpox.
ESTHER : Lustig_2009_Vaccine_27_1691
PubMedSearch : Lustig_2009_Vaccine_27_1691
PubMedID: 19195492

Title : Accommodation of physostigmine and its analogues by acetylcholinesterase is dominated by hydrophobic interactions - Barak_2009_Biochem.J_417_213
Author(s) : Barak D , Ordentlich A , Stein D , Yu QS , Greig NH , Shafferman A
Ref : Biochemical Journal , 417 :213 , 2009
Abstract : The role of the functional architecture of the HuAChE (human acetylcholinesterase) in reactivity toward the carbamates pyridostigmine, rivastigmine and several analogues of physostigmine, that are currently used or considered for use as drugs for Alzheimer's disease, was analysed using over 20 mutants of residues that constitute the interaction subsites in the active centre. Both steps of the HuAChE carbamylation reaction, formation of the Michaelis complex as well as the nucleophilic process, are sensitive to accommodation of the ligand by the enzyme. For certain carbamate/HuAChE combinations, the mode of inhibition shifted from a covalent to a noncovalent type, according to the balance between dissociation and covalent reaction rates. Whereas the charged moieties of pyridostigmine and rivastigmine contribute significantly to the stability of the corresponding HuAChE complexes, no such effect was observed for physostigmine and its analogues, phenserine and cymserine. Moreover, physostigmine-like ligands carrying oxygen instead of nitrogen at position -1 of the tricyclic moiety (physovenine and tetrahydrofurobenzofuran analogues) displayed comparable structure-function characteristics toward the various HuAChE enzymes. The essential role of the HuAChE hydrophobic pocket, comprising mostly residues Trp(86) and Tyr(337), in accommodating (-)-physostigmine and in conferring approximately 300-fold stereoselectivity toward physostigmines, was elucidated through examination of the reactivity of selected HuAChE mutations toward enantiomeric pairs of different physostigmine analogues. The present study demonstrates that certain charged and uncharged ligands, like analogues of physostigmine and physovenine, seem to be accommodated by the enzyme mostly through hydrophobic interactions.
ESTHER : Barak_2009_Biochem.J_417_213
PubMedSearch : Barak_2009_Biochem.J_417_213
PubMedID: 18729824

Title : Isolation and chimerization of a highly neutralizing antibody conferring passive protection against lethal Bacillus anthracis infection - Rosenfeld_2009_PLoS.One_4_e6351
Author(s) : Rosenfeld R , Marcus H , Ben-Arie E , Lachmi BE , Mechaly A , Reuveny S , Gat O , Mazor O , Ordentlich A
Ref : PLoS ONE , 4 :e6351 , 2009
Abstract : Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD(50)B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD(50) of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax.
ESTHER : Rosenfeld_2009_PLoS.One_4_e6351
PubMedSearch : Rosenfeld_2009_PLoS.One_4_e6351
PubMedID: 19629185

Title : Flexibility versus rigidity of the functional architecture of AChE active center - Shafferman_2008_Chem.Biol.Interact_175_166
Author(s) : Shafferman A , Barak D , Stein D , Kronman C , Velan B , Greig NH , Ordentlich A
Ref : Chemico-Biological Interactions , 175 :166 , 2008
Abstract : Functional architecture of the AChE active center appears to be characterized by both structural "rigidity", necessary to stabilize the catalytic triad as well as by flexibility in accommodating the different, high affinity AChE ligands. These seemingly conflicting structural properties of the active center are demonstrated through combination of structural methods with kinetic studies of the enzyme and its mutant derivatives with plethora of structurally diverse ligands and in particular with series of stereoselective covalent and noncovalent AChE ligands. Thus, steric perturbation of the acyl pocket precipitates in a pronounced stereoselectivity toward methylphosphonates by disrupting the stabilizing environment of the catalytic histidine rather than through steric exclusion demonstrating the functional importance of the "rigid" environment of the catalytic machinery. The acyl pocket, the cation-binding subsite (Trp86) and the peripheral anionic subsite were also found to be directly involved in HuAChE stereoselectivity toward charged chiral phosphonates, operating through differential positioning of the ligand cationic moiety within the active center. Residue Trp86 is also a part of the "hydrophobic patch" which seems flexible enough to accommodate the structurally diverse ligands like tacrine, galanthamine and the two diastereomers of huperzine A. Also, we have recently discovered further aspects of the role of both the unique structure and the flexibility of the "hydrophobic patch" in determining the reactivity and stereoselectivity of HuAChE toward certain carbamates including analogs of physostigmine. In these cases the ligands are accommodated mostly through hydrophobic interactions and their stereoselectivity delineates precisely the steric limits of the pocket. Hence, the HuAChE stereoselectivity provides a sensitive tool in the in depth exploration of the functional architecture of the active center. These studies suggest that the combination of "rigidity" and flexibility within the HuAChE gorge are an essential element of its molecular design.
ESTHER : Shafferman_2008_Chem.Biol.Interact_175_166
PubMedSearch : Shafferman_2008_Chem.Biol.Interact_175_166
PubMedID: 18471807

Title : Aging-resistant organophosphate bioscavenger based on polyethylene glycol-conjugated F338A human acetylcholinesterase - Mazor_2008_Mol.Pharmacol_74_755
Author(s) : Mazor O , Cohen O , Kronman C , Raveh L , Stein D , Ordentlich A , Shafferman A
Ref : Molecular Pharmacology , 74 :755 , 2008
Abstract : The high reactivity of cholinesterases (ChEs) toward organophosphorus (OP) compounds has led to propose recombinant ChEs as bioscavengers of nerve agents. The bioscavenging potential of recombinant ChEs can be enhanced by conjugation of polyethylene glycol (PEG) moieties, to extend their circulatory residence. However, the ability of exogenously administered ChEs to confer long-term protection against repeated exposures to nerve agents is still limited due to the aging process, whereby organophosphate-ChE adducts undergo irreversible dealkylation, which precludes oxime-mediated reactivation of the enzyme. To generate an optimal acetylcholinesterase (AChE)-based OP bioscavenger, the F338A mutation, known to decelerate the rate of aging of AChE-OP conjugates, was incorporated into polyethylene glycol-conjugated (PEGylated) human AChE. The PEGylated F338A-AChE displayed unaltered rates of hydrolysis, inhibition, phosphylation, and reactivation and could effectively protect mice against exposure to soman (pinacolylmethyl phosphonofluoridate), sarin (O-isopropyl methylphosphonofluoridate), or O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothioate (VX). Unlike PEGylated wild-type (WT)-AChE, the PEGylated F338A-AChE exhibits significantly reduced aging rates after soman inhibition and can be efficiently reactivated by the 1-[[[4(aminocarbonyl)-pyridinio]methoxy]methyl]-2(hydroxyimino)methyl]pyridinium dichloride (HI-6) oxime, both in vitro and in vivo. Accordingly, oxime administration to PEG-F338A-AChE-pretreated mice enabled them to withstand repeated soman exposure (5.4 and 4 LD(50)/dose), whereas same regime treatment of non-PEGylated F338A-AChE- or PEGylated WT-AChE-pretreated mice failed to protect against the second challenge, due to rapid clearance or irreversible aging of the latter enzymes. Thus, judicious incorporation of selected mutations into the AChE mold in conjunction with its chemical modification provides means to engineer an optimal ChE-based OP bioscavenger in terms of circulatory longevity, resistance to aging, and reduced doses required for protection, even against repeated exposures to nerve agents, such as soman.
ESTHER : Mazor_2008_Mol.Pharmacol_74_755
PubMedSearch : Mazor_2008_Mol.Pharmacol_74_755
PubMedID: 18523134

Title : Polyethylene-glycol conjugated recombinant human acetylcholinesterase serves as an efficacious bioscavenger against soman intoxication - Kronman_2007_Toxicology_233_40
Author(s) : Kronman C , Cohen O , Raveh L , Mazor O , Ordentlich A , Shafferman A
Ref : Toxicology , 233 :40 , 2007
Abstract : Extensive pharmacokinetic studies in both mice and rhesus macaques, with biochemically well defined forms of native and recombinant AChEs from bovine, rhesus and human origin, allowed us to determine an hierarchical pattern by which post-translation-related factors and specific amino-acid epitopes govern the pharmacokinetic performance of the enzyme molecule. In parallel, we demonstrated that controlled conjugation of polyethylene-glycol (PEG) side-chains to lysine residues of rHuAChE also results in the generation of active enzyme with improved pharmacokinetic performance. Here, we show that equally efficient extension of circulatory residence can be achieved by specific conditions of PEGylation, regardless of the post-translation-modification state of the enzyme. The masking effect of PEGylation, which is responsible for extending circulatory lifetime, also contributes to the elimination of immunological responses following repeated administration of AChE. Finally, in vivo protection studies in mice allowed us to determine that the PEGylated AChE protects the animal from a high lethal dose (2.5 LD(50)) of soman. On a mole basis, both the recombinant AChE and its PEGylated form provide higher levels of protection against soman poisoning than the native serum-derived HuBChE. The findings that circulatory long-lived PEGylated AChE can confer superior protection to mice against OP-compound poisoning while exhibiting reduced immunogenicity, suggest that this chemically modified version of rHuAChE may serve as a highly effective bioscavenger for prophylactic treatment against OP-poisoning.
ESTHER : Kronman_2007_Toxicology_233_40
PubMedSearch : Kronman_2007_Toxicology_233_40
PubMedID: 17045722

Title : Comparison of polyethylene glycol-conjugated recombinant human acetylcholinesterase and serum human butyrylcholinesterase as bioscavengers of organophosphate compounds - Cohen_2006_Mol.Pharmacol_70_1121
Author(s) : Cohen O , Kronman C , Raveh L , Mazor O , Ordentlich A , Shafferman A
Ref : Molecular Pharmacology , 70 :1121 , 2006
Abstract : Comparative protection studies in mice demonstrate that on a molar basis, recombinant human acetylcholinesterase (rHuAChE) confers higher levels of protection than native human butyrylcholinesterase (HuBChE) against organophosphate (OP) compound intoxication. For example, mice challenged with 2.5 LD50 of O-isopropyl methylphosphonofluoridate (sarin), pinacolylmethyl phosphonofluoridate (soman), and O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothiolate (VX) after treatment with equimolar amounts of the two cholinesterases displayed 80, 100, and 100% survival, respectively, when pre-treatment was carried out with rHuAChE and 0, 20, and 60% survival, respectively, when pretreatment was carried out with HuBChE. Kinetic studies and active site titration analyses of the tested OP compounds with acetylcholinesterases (AChEs) and butyrylcholinesterases (BChEs) from different mammalian species demonstrate that the superior in vivo efficacy of acetyl-cholinesterases is in accordance with the higher stereoselectivity of AChE versus BChE toward the toxic enantiomers comprising the racemic mixtures of the various OP agents. In addition, we show that polyethylene glycol-conjugated (PEGy-lated) rHuAChE, which is characterized by a significantly extended circulatory residence both in mice and monkeys ( Biochem J 357: 795-802, 2001 ; Biochem J 378: 117-128, 2004 ), retains full reactivity toward OP compounds both in vitro and in vivo and provides a higher level of protection to mice against OP poisoning, compared with native serum-derived HuBChE. Indeed, PEGylated rHuAChE also confers superior prophylactic protection when administered intravenously or intramuscularly over 20 h before exposure of mice to a lethal dose of VX (1.3-1.5 LD50). These findings together with the observations that the PEGylated rHuAChE exhibits unaltered biodistribution and high bioavailability present a case for using PEGylated rHuAChE as a very efficacious bioscavenger of OP agents.
ESTHER : Cohen_2006_Mol.Pharmacol_70_1121
PubMedSearch : Cohen_2006_Mol.Pharmacol_70_1121
PubMedID: 16801396

Title : The role of AChE active site gorge in determining stereoselectivity of charged and noncharged VX enantiomers - Ordentlich_2005_Chem.Biol.Interact_157-158_191
Author(s) : Ordentlich A , Barak D , Sod-Moriah G , Kaplan D , Mizrahi D , Segall Y , Kronman C , Karton Y , Lazar A , Marcus D , Velan B , Shafferman A
Ref : Chemico-Biological Interactions , 157-158 :191 , 2005
Abstract : The reactivity of human acetylcholinesterase (HuAChE) toward the chemical warfare agent VX [O-ethyl S-[2-(diisopropylamino)ethyl] methyl-phosphonothioate] and its stereoselectivity toward the P(S)-enantiomer were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere nc-VX [O-ethyl S-(3-isopropyl-4-methyl-pentyl) methylphosphonothioate]. Stereoselectivity of the wild-type HuAChE toward VX(S) is manifested by a 115-fold higher bimolecular rate constant (1.4 x 10(8) min(-1) M(-1)) as compared to that of VX(R). HuAChE was also 12,500-fold more reactive toward VX(S) than toward nc-VX(S), demonstrating the significance of the polar interactions of the ammonium substituent to their overall affinity toward VX. Indeed, substitution of the cation-binding subsite residue Trp86 by alanine resulted in a decrease of three orders of magnitude in HuAChE reactivity toward both VX enantiomers, with only a marginal effect on the reactivity toward the enantiomers of nc-VX. These results demonstrate that accommodation of the charged moieties of both VX enantiomers depends predominantly on interactions with the aromatic moiety of Trp86. Yet, these interactions seem to limit the stereoselectivity toward the P(S)-enantiomer, which for charged methylphosphonates is much lower than for the noncharged analogs, like sarin or soman. Marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 by asparagine (D74N) practically abolished stereoselectivity toward VX(S) (a 130-fold decrease), while substitution which retained the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers originates predominantly from a different orientation of the charged leaving group with respect to residue Asp74. Such different orientations of the charged leaving group in the HuAChE adducts of the VX enantiomers seem to be a consequence of intramolecular interactions with the bulky phosphorus alkoxy group.
ESTHER : Ordentlich_2005_Chem.Biol.Interact_157-158_191
PubMedSearch : Ordentlich_2005_Chem.Biol.Interact_157-158_191
PubMedID: 16289014

Title : Functional requirements for the optimal catalytic configuration of the AChE active center - Shafferman_2005_Chem.Biol.Interact_157-158_123
Author(s) : Shafferman A , Barak D , Kaplan D , Ordentlich A , Kronman C , Velan B
Ref : Chemico-Biological Interactions , 157-158 :123 , 2005
Abstract : Functional analysis of the HuAChE active center architecture revealed that accommodation of structurally diverse substrates and other ligands is achieved through interactions with specific subsites such as the acyl pocket, cation binding site, hydrophobic site or the oxyanion hole. Recent studies have begun to unravel the role of this active center architecture in maintaining the optimal catalytic facility of the enzyme through inducing proper alignment of the catalytic triad. The exact positioning of the catalytic glutamate (Glu334) seems to be determined by a hydrogen bond network including several polar residues and water molecules. Disruption of this network by replacement of Ser229 by alanine is thought to remove the Glu334 carboxylate from the vicinity of His447 abolishing catalytic activity. The proper orientation of the catalytic histidine side chain is maintained by these polar interactions as well as through "aromatic trapping" by residues lining the HuAChE active center gorge. Thus, replacement of aromatic residues in the vicinity of His447, as in the F295A/F338A or in the Y72N/Y124Q/W286A/F295L/F297V/Y337A (hexamutant which mimicks the aromatic lining of HuBChE) enzymes, resulted in a dramatic decrease in catalytic activity, which was proposed to originate from catalytically nonproductive mobility of His447. Yet, HuBChE is catalytically efficient indicating that "aromatic trapping" is not the only way to conformationally stabilize the His447 side chain. A possible restriction of this mobility in a series of F295X/F338A HuAChEs was examined in silico followed by site-directed mutagenesis. Both simulations and reactivities of the actual F295X/F338A enzymes, carrying various aliphatic residues at position 295, indicate that of the bulky amino acids, like leucine or isoleucine, only methionine was capable of maintaining the catalytically viable conformation of His447. The F295M/F338A HuAChE was only two-fold less reactive than the F338A enzyme toward acetylthiocholine, and exhibited wild type-like reactivity toward covalent modifiers of the catalytic Ser203. The findings are consistent with the notion that different combinations of steric interference and specific polar interactions serve to maintain the position of His447 and thereby the high efficiency of the catalytic machinery. The two seemingly conflicting demands on the architecture of the active center-flexible accommodation of substrate and optimal juxtaposition of residues of the catalytic triad, demonstrate the truly amazing molecular design of the AChE active center.
ESTHER : Shafferman_2005_Chem.Biol.Interact_157-158_123
PubMedSearch : Shafferman_2005_Chem.Biol.Interact_157-158_123
PubMedID: 16256968

Title : Lessons from functional analysis of AChE covalent and noncovalent inhibitors for design of AD therapeutic agents - Barak_2005_Chem.Biol.Interact_157-158_219
Author(s) : Barak D , Ordentlich A , Kaplan D , Kronman C , Velan B , Shafferman A
Ref : Chemico-Biological Interactions , 157-158 :219 , 2005
Abstract : Determination of the 3D-structure of acetylcholinesterase (AChE) of Torpedo californica over a decade ago, and more recently that of human enzyme together with extensive targeted mutagenesis of the mammalian AChEs led to a fine mapping of the multiple functional domains within the active center of the enzyme. Many of the contributions of this active center architecture to accommodation of noncovalent ligands could be deduced from the X-ray structures of the corresponding HuAChE complexes. Yet, Michaelis complexes leading to transient covalent adducts are not amenable to structural analysis. Since the rates of formation of the covalent adducts depend predominantly on the stabilities of the corresponding Michaelis complexes, it is essential to characterize the specific interactions contributing to stabilization of these complexes. Functional analysis of interactions with HuAChE enzymes allows for such characterization for carbamates, like pyridostigmine or rivastigmine, much in the same way as that for the noncovalent therapeutic ligands nivalin or aricept. In fact, the observed differences between the affinities toward carbamates and the noncovalent ligands seem to result from specific structural characteristics of the inhibitors rather than from the decomposition path of the particular complex. Replacements at the cation binding site (Trp86), hydrogen bond network (Glu202, Tyr133, Glu450), and hydrophobic pocket result in similar effects for the covalent as well as for the noncovalent inhibitors. Also, while the effects of perturbing the aromatic trapping of the catalytic His447 for pyridostigmine and nivalin were analogous to those for the substrate, the corresponding effects for rivastigmine and aricept were quite different. Thus, elucidation of the functional architecture of the HuAChE active center is bound to be of considerable utility in the current effort to design novel covalent AChE inhibitors as therapeutics for Alzheimer's disease (AD).
ESTHER : Barak_2005_Chem.Biol.Interact_157-158_219
PubMedSearch : Barak_2005_Chem.Biol.Interact_157-158_219
PubMedID: 16289124

Title : Poster (35) The hierarchy of post-translation modifications determining circulatory longevity of acetylcholinesterase. -
Author(s) : Velan B , Kronman C , Chitlaru T , Cohen O , Ordentlich A , Shafferman A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :339 , 2004
PubMedID:

Title : Surprising findings from the functional analysis of human acetylcholinesterase adducts of Alzheimer?s disease drugs. -
Author(s) : Ordentlich A , Barak D , Ariel N , Kronman C , Kaplan D , Velan B , Shafferman A
Ref : Cholinergic Mechanisms, CRC Press :177 , 2004
PubMedID:

Title : Generation of pharmacokinetically improved recombinant human acetylcholinesterase by polyethylene glycol modification. -
Author(s) : Cohen O , Kronman C , Chitlaru T , Ordentlich A , Velan B , Shafferman A
Ref : Cholinergic Mechanisms, CRC Press :519 , 2004
PubMedID:

Title : Attempts to engineer an enzyme mimic of butyrylcholinesterase by substitution of the six divergent aromatic amino acids in the active center of acetylcholinesterase. -
Author(s) : Kaplan D , Ordentlich A , Barak D , Ariel N , Kronman C , Baruch V , Shafferman A
Ref : Cholinergic Mechanisms, CRC Press :601 , 2004
PubMedID:

Title : A complex array of post-translation modifications determines the circulatory longevity of acetylcholinesterase in a hierarchical manner. -
Author(s) : Shafferman A , Chitlaru T , Ordentlich A , Velan B , Kronman C
Ref : Cholinergic Mechanisms, CRC Press :245 , 2004
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Title : MALDI-TOF\/MS analysis of tabun-acetylcholinesterase conjugate. -
Author(s) : Elhanany E , Ordentlich A , Dgany OR , Kaplan D , Segall Y , Barak R , Velan B , Shafferman A
Ref : Cholinergic Mechanisms, CRC Press :563 , 2004
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Title : Poster (14) The aromatic trapping of histidine 447 in catalysis of acetylcholinesterases -
Author(s) : Shafferman A , Barak D , Kaplan D , Ordentlich A , Ariel N , Velan B
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :327 , 2004
PubMedID:

Title : The aromatic trapping of histidine 447 in catalysis of acetylcholinesterases -
Author(s) : Shafferman A , Barak D , Kaplan D , Ordentlich A , Ariel N , Velan B
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :181 , 2004
PubMedID:

Title : Stereoselectivity toward VX is determined by interactions with residues of the acyl pocket as well as of the peripheral anionic site of AChE - Ordentlich_2004_Biochemistry_43_11255
Author(s) : Ordentlich A , Barak D , Sod-Moriah G , Kaplan D , Mizrahi D , Segall Y , Kronman C , Karton Y , Lazar A , Marcus D , Velan B , Shafferman A
Ref : Biochemistry , 43 :11255 , 2004
Abstract : The origins of human acetylcholinesterase (HuAChE) reactivity toward the lethal chemical warfare agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) and its stereoselectivity toward the P(S)-VX enantiomer (VX(S)) were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere O-ethyl S-(3-isopropyl-4-methylpentyl) methylphosphonothioate (nc-VX) as well as echothiophate and its noncharged analogue. Reactivity of wild-type HuAChE toward VX(S) was 115-fold higher than that toward VX(R), with bimolecular rate constants of 1.4 x 10(8) and 1.2 x 10(6) min(-1) M(-1). HuAChE was also 12500-fold more reactive toward VX(S) than toward nc-VX(S). Substitution of the cation binding subsite residue Trp86 with alanine resulted in a 3 order of magnitude decrease in HuAChE reactivity toward both VX enantiomers, while this replacement had an only marginal effect on the reactivity toward the enantiomers of nc-VX and the noncharged echothiophate. These results attest to the critical role played by Trp86 in accommodating the charged moieties of both VX enantiomers. A marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 with asparagine (D74N) practically abolished stereoselectivity toward VX(S) (130-fold decrease), while a substitution which retains the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers is mainly a result of a different interaction of the charged leaving group with Asp74.
ESTHER : Ordentlich_2004_Biochemistry_43_11255
PubMedSearch : Ordentlich_2004_Biochemistry_43_11255
PubMedID: 15366935

Title : Poster (21) Analysis of acetylcholinesterase adducts of alzheimer's drugs -
Author(s) : Ordentlich A , Kronman C , Barak D , Ariel N , Kaplan D , Velan B , Shafferman A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :332 , 2004
PubMedID:

Title : Poster (23) Phosphorylation and aging of AChE - insights from mutagenesis, mass spectrometry and structural studies -
Author(s) : Barak D , Ordentlich A , Kaplan D , Elhanani E , Segall Y , Barak R , Velan B , Shafferman A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :332 , 2004
PubMedID:

Title : Is aromaticity essential for trapping the catalytic histidine 447 in human acetylcholinesterase? - Kaplan_2004_Biochemistry_43_3129
Author(s) : Kaplan D , Barak D , Ordentlich A , Kronman C , Velan B , Shafferman A
Ref : Biochemistry , 43 :3129 , 2004
Abstract : Replacement of both the acyl pocket residue Phe295 as well as residue Phe338, adjacent to the catalytic His447 in human acetylcholinesterase (HuAChE), resulted in a 680-fold decline in catalytic activity due to conformational destabilization of the histidine side chain [Barak et al. (2002) Biochemistry 41, 8245]. A possible restriction of this catalytically nonproductive mobility of His447 in a series of F295X/F338A HuAChEs was examined in silico followed by site-directed mutagenesis. Simulations suggested that of the 12 aliphatic residues substituted at position 295, including hydrophobic and polar amino acids, only methionine was capable of maintaining the catalytically viable conformation of His447. Examination of the reactivities of the actual F295X/F338A HuAChEs showed that indeed the F295M/F338A enzyme was only 2-fold less reactive than the F338A mutant toward acetylthiocholine, while enzymes substituted by the similarly bulky residues leucine and isoleucine were catalytically impaired. Furthermore, only the F295M/F338A enzyme exhibited wild-type-like reactivity toward covalent modifiers of the catalytic Ser203 including the methylphosphonate soman and transition state analogue m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), as well as a facile dealkylation of the F295M/F338A-soman adduct. A different behavior was observed for bulkier ligands which introduce a deformation in the acyl pocket, and therefore their activity seems only marginally affected by the positioning of His447. The findings emphasize the importance of the precise positioning of His447 for catalysis and indicate that, in the absence of aromatic "trapping", restriction of the histidine mobility in F295X/F338A HuAChEs requires a combination of steric interference and a specific polar interaction. The results also underscore the role of the acyl pocket subsite of cholinesterases in maintaining the catalytically viable conformation of the catalytic histidine.
ESTHER : Kaplan_2004_Biochemistry_43_3129
PubMedSearch : Kaplan_2004_Biochemistry_43_3129
PubMedID: 15023064

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 : The aromatic trapping of the catalytic histidine is essential for efficient catalysis in acetylcholinesterase - Barak_2002_Biochemistry_41_8245
Author(s) : Barak D , Kaplan D , Ordentlich A , Ariel N , Velan B , Shafferman A
Ref : Biochemistry , 41 :8245 , 2002
Abstract : While substitution of the aromatic residues (Phe295, Phe338), located in the vicinity of the catalytic His447 in human acetylcholinesterase (HuAChE) had little effect on catalytic activity, simultaneous replacement of both residues by aliphatic amino acids resulted in a 680-fold decrease in catalytic activity. Molecular simulations suggested that the activity decline is related to conformational destabilization of His447, similar to that observed for the hexamutant HuAChE which mimics the active center of butyrylcholinesterase. On the basis of model structures of other cholinesterases (ChEs), we predicted that catalytically nonproductive mobility of His447 could be restricted by introduction of aromatic residue in a different location adjacent to this histidine (Val407). Indeed, the F295A/F338A/V407F enzyme is 170-fold more reactive than the corresponding double mutant and only 3-fold less reactive than the wild-type HuAChE. However, analogous substitution of Val407 in the hexamutant HuAChE (generating the heptamutant Y72N/Y124Q/W286A/F295L/F297V/Y337A/V407F) did not enhance catalytic activity. Reactivity of these double, triple, hexa, and hepta mutant HuAChEs was monitored toward covalent ligands such as organophosphates and the transition state analogue TMFTA, which probe, respectively, the facility of the enzymes to accommodate Michaelis complexes and to undergo the acylation process. The findings suggest that in the F295A/F338A mutant the two His447 conformational states, which are essential for the different stages of the catalytic process, seem to be destabilized. On the other hand, in the F295A/F338A/V407F mutant only the state involved in acylation is impaired. Such differential effects on the His447 conformational properties demonstrate the general role of aromatic residues in cholinesterases, and probably in other serine hydrolases, in "trapping" of the catalytic histidine and thereby in optimization of catalytic activity.
ESTHER : Barak_2002_Biochemistry_41_8245
PubMedSearch : Barak_2002_Biochemistry_41_8245
PubMedID: 12081473

Title : Effect of chemical modification of recombinant human acetylcholinesterase by polyethylene glycol on its circulatory longevity - Cohen_2001_Biochem.J_357_795
Author(s) : Cohen O , Kronman C , Chitlaru T , Ordentlich A , Velan B , Shafferman A
Ref : Biochemical Journal , 357 :795 , 2001
Abstract : Post-translational modifications were recently shown to be responsible for the short circulatory mean residence time (MRT) of recombinant human acetylcholinesterase (rHuAChE) [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959--967; Chitlaru, Kronman, Zeevi, Kam, Harel, Ordentlich, Velan and Shafferman (1998) Biochem. J. 336, 647--658; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613--625], which is one of the major obstacles to the fulfilment of its therapeutic potential as a bioscavenger. In the present study we demonstrate that the MRT of rHuAChE can be significantly increased by the controlled attachment of polyethylene glycol (PEG) side chains to lysine residues. Attachment of as many as four PEG molecules to monomeric rHuAChE had minimal effects, if any, on either the catalytic activity (K(m)=0.09 mM and k(cat)=3.9 x 10(5) min(-1)) or the reactivity of the modified enzyme towards active-centre inhibitors, such as edrophonium and di-isopropyl fluorophosphate, or to peripheral-site ligands, such as propidium, BW284C51 and even the bulky snake-venom toxin fasciculin-II. The increase in MRT of the PEG-modified monomeric enzyme is linearly dependent, in the tested range, on the number of attached PEG molecules, as well as on their size. It appears that even low level PEG-conjugation can overcome the deleterious effect of under-sialylation on the pharmacokinetic performance of rHuAChE. At the highest tested ratio of attached PEG-20000/rHuAChE (4:1), an MRT of over 2100 min was attained, a value unmatched by any other known form of recombinant or native serum-derived AChE reported to date.
ESTHER : Cohen_2001_Biochem.J_357_795
PubMedSearch : Cohen_2001_Biochem.J_357_795
PubMedID: 11463350

Title : Does butyrylization of acetylcholinesterase through substitution of the six divergent aromatic amino acids in the active center gorge generate an enzyme mimic of butyrylcholinesterase? - Kaplan_2001_Biochemistry_40_7433
Author(s) : Kaplan D , Ordentlich A , Barak D , Ariel N , Kronman C , Velan B , Shafferman A
Ref : Biochemistry , 40 :7433 , 2001
Abstract : The active center gorge of human acetylcholinesterase (HuAChE) is lined by 14 aromatic residues, whereas in the closely related human butyrylcholinesterase (HuBChE) 3 of the aromatic active center residues (Phe295, Phe297, Tyr337) as well as 3 of the residues at the gorge entrance (Tyr72, Tyr124, Trp286) are replaced by aliphatic amino acids. To investigate whether this structural variability can account for the reactivity differences between the two enzymes, gradual replacement of up to all of the 6 aromatic residues in HuAChE by the corresponding residues in HuBChE was carried out. The affinities of the hexamutant (Y72N/Y124Q/W286A/F295L/F297V/Y337A) toward tacrine, decamethonium, edrophonium, huperzine A, or BW284C51 differed by about 5-, 80-, 170-, 25000-, and 17000-fold, respectively, from those of the wild-type HuAChE. For most of these prototypical noncovalent active center and peripheral site ligands, the hexamutant HuAChE displayed a reactivity phenotype closely resembling that of HuBChE. These results support the accepted view that the active center architectures of AChE and BChE differ mainly by the presence of a larger void space in BChE. Nevertheless, reactivity of the hexamutant HuAChE toward the substrates acetylthiocholine and butyrylthiocholine, or covalent ligands such as phosphonates and the transition state analogue m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), is about 45-170-fold lower than that of HuBChE. Most of this reduction in reactivity can be related to the combined replacements of the three aromatic residues at the active center, Phe295, Phe297, and Tyr337. We propose that the hexamutant HuAChE, unlike BChE, is impaired in its capacity to accommodate certain tetrahedral species in the active center. This impairment may be related to the enhanced mobility of the catalytic histidine His447, which is observed in molecular dynamics simulations of the hexamutant and the F295L/F297V/Y337A HuAChE enzymes but not in the wild-type HuAChE.
ESTHER : Kaplan_2001_Biochemistry_40_7433
PubMedSearch : Kaplan_2001_Biochemistry_40_7433
PubMedID: 11412096

Title : Resolving pathways of interaction of covalent inhibitors with the active site of acetylcholinesterases: maldi-tof\/ms analysis of various nerve agent phosphyl adducts - Elhanany_2001_Chem.Res.Toxicol_14_912
Author(s) : Elhanany E , Ordentlich A , Dgany O , Kaplan D , Segall Y , Barak R , Velan B , Shafferman A
Ref : Chemical Research in Toxicology , 14 :912 , 2001
Abstract : Understanding reaction pathways of phosphylation, reactivation, and "aging" of AChE with toxic organophosphate compounds is both a biochemical and a pharmacological challenge. Here we describe experiments which allowed to resolve some of the less well understood reaction pathways of phosphylation and "aging" of acetylcholinesterase (AChE) involving phosphoroamidates (P-N agents) such as tabun or the widely used pesticide methamidophos. Tryptic digests of phosphylated AChEs (from human and Torpedo californica), ZipTip peptide fractionation and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) enabled reproducible signal enrichment of the isotopically resolved peaks of organophosphoroamidate conjugates of the AChE active site Ser peptides. For tabun and its hexadeuterio analogue, we find, as expected, that the two phosphoramidate adducts of the active site peptide differ by 6.05 mass units but following aging we find that the two corresponding phospho-peptides have identical molecular weights. We further show that the aging product of paraoxon-AChE adduct is identical to the aging product of the tabun-AChE conjugate. These results unequivocally demonstrate that the pathway of aging of tabun adducts of the human or the Torpedo californica AChEs proceeds through P-N bond scission. For methamidophos, we show that phosphylation of AChE involves elimination of the thiomethyl moiety and that the spontaneous reactivation of the resulting organophosphate adduct generates the phosphorus free AChE active site Ser-peptide.
ESTHER : Elhanany_2001_Chem.Res.Toxicol_14_912
PubMedSearch : Elhanany_2001_Chem.Res.Toxicol_14_912
PubMedID: 11453739

Title : Evidence for P-N bond scission in phosphoroamidate nerve agent adducts of human acetylcholinesterase - Barak_2000_Biochemistry_39_1156
Author(s) : Barak D , Ordentlich A , Kaplan D , Barak R , Mizrahi D , Kronman C , Segall Y , Velan B , Shafferman A
Ref : Biochemistry , 39 :1156 , 2000
Abstract : Acetylcholinesterases (AChEs) form conjugates with certain highly toxic organophosphorus (OP) agents that become gradually resistant to reactivation. This phenomenon termed "aging" is a major factor limiting the effectiveness of therapy in certain cases of OP poisoning. While AChE adducts with phosphonates and phosphates are known to age through scission of the alkoxy C-O bond, the aging path for adducts with phosphoroamidates (P-N agents) like the nerve agent N,N-dimethylphosphonocyanoamidate (tabun) is not clear. Here we report that conjugates of tabun and of its butyl analogue (butyl-tabun) with the E202Q and F338A human AChEs (HuAChEs) age at similar rates to that of the wild-type enzyme. This is in marked contrast to the large effect of these substitutions on the aging of corresponding adducts with phosphates and phosphonates, suggesting that a different aging mechanism may be involved. Both tabun and butyl-tabun appear to be similarly accommodated in the active center, as suggested by molecular modeling and by kinetic studies of phosphylation and aging with a series of HuAChE mutants (E202Q, F338A, F295A, F297A, and F295L/F297V). Mass spectrometric analysis shows that HuAChE adduct formation with tabun and butyl-tabun occurs through loss of cyanide and that during the aging process both of these adducts show a mass decrease of 28 +/- 4 Da. Due to the nature of the alkoxy substituent, such mass decrease can be unequivocally assigned to loss of the dimethylamino group, at least for the butyl-tabun conjugate. This is the first demonstration that AChE adducts with toxic P-N agents can undergo aging through scission of the P-N bond.
ESTHER : Barak_2000_Biochemistry_39_1156
PubMedSearch : Barak_2000_Biochemistry_39_1156
PubMedID: 10653663

Title : Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level - Millard_1999_Biochemistry_38_7032
Author(s) : Millard CB , Kryger G , Ordentlich A , Greenblatt HM , Harel M , Raves ML , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
Ref : Biochemistry , 38 :7032 , 1999
Abstract : Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 A resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine. Phosphorylation with DFP caused an unexpected movement in the main chain of a loop that includes residues F288 and F290 of the TcAChE acyl pocket. This is the first major conformational change reported in the active site of any AChE-ligand complex, and it offers a structural explanation for the substrate selectivity of AChE.
ESTHER : Millard_1999_Biochemistry_38_7032
PubMedSearch : Millard_1999_Biochemistry_38_7032
PubMedID: 10353814
Gene_locus related to this paper: torca-ACHE

Title : Reaction Products of Acetylcholinesterase and Vx Reveal a Mobile Histidine in the Catalytic Triad - Millard_1999_J.Am.Chem.Soc_121_9883
Author(s) : Millard CB , Koellner G , Ordentlich A , Shafferman A , Silman I , Sussman JL
Ref : Journal of the American Chemical Society , 121 :9883 , 1999
Abstract : The presence of a precisely aligned active-site triad (Ser-His-Asp/Glu) in the three-dimensional structures of widely different hydrolytic enzymes has generated intense interest in the chemical modus operandi of this catalytic motif. 1 One hypothesis, which has not received wide acceptance, proposes that the imidazole of the catalytic His is mobile during enzyme function. 2 We solved the structures of the phosphonylation and dealkylation ("aging") reaction products of acetylcholinesterase (AChE; EC 3.1.1.7) and an organophosphorus (OP) inhibitor, O-ethyl-S-[2-[bis(1-meth-ylethyl) amino]ethyl] methylphosphonothioate (VX) by X-ray crystallography. The structures clearly demonstrate reversible movement of the catalytic His. Moreover, the conformational change apparently involves a hydrogen (H-) bond with a glutamate (E199) which had been implicated previously in OP and substrate reactions.
ESTHER : Millard_1999_J.Am.Chem.Soc_121_9883
PubMedSearch : Millard_1999_J.Am.Chem.Soc_121_9883
PubMedID:
Gene_locus related to this paper: torca-ACHE

Title : A preliminary comparison of structural models for catalytic intermediates of acetylcholinesterase - Silman_1999_Chem.Biol.Interact_119-120_43
Author(s) : Silman I , Millard CB , Ordentlich A , Greenblatt HM , Harel M , Barak D , Shafferman A , Sussman JL
Ref : Chemico-Biological Interactions , 119-120 :43 , 1999
Abstract : Determination of the three dimensional structure of Torpedo Californica acetylcholinesterase (TcAChE) provided an experimental tool for directly visualizing interaction of AChE with cholinesterase inhibitors of fundamental, pharmacological and toxicological interest. The structure revealed that the active site is located near the bottom of a deep and narrow gorge lined with 14 conserved aromatic amino acids. The structure of a complex of TcAChE with the powerful 'transition state analog' inhibitor, TMTFA, suggested that its orientation in the experimentally determined structure was very similar to that proposed for the natural substrate, acetylcholine, by manual docking. The array of enzyme-ligand interactions visualized in the TMFTA complex also are expected to envelope the unstable TI that forms with acetylcholine during acylation, and to sequester it from solvent. In our most recent studies, the crystal structures of several 'aged' conjugates of TcAChE obtained with OP nerve agents have been solved and compared with that of the native enzyme. The methylphosphonylated-enzyme obtained by reaction with soman provides a useful structural analog for the TI that forms during deacylation after the reaction of TcAChE with acetylcholine. By comparing these structures, we conclude that the same 'oxyanion hole' residues, as well as the aromatic side chains constituting the 'acyl pocket', participate in acylation (TMTFA-AChE) and deacylation (OP-AChE), and that AChE can accommodate both TIs at the bottom of the gorge without major conformational movements.
ESTHER : Silman_1999_Chem.Biol.Interact_119-120_43
PubMedSearch : Silman_1999_Chem.Biol.Interact_119-120_43
PubMedID: 10421437
Gene_locus related to this paper: torca-ACHE

Title : Exploring the active center of human acetylcholinesterase with stereomers of an organophosphorus inhibitor with two chiral centers - Ordentlich_1999_Biochemistry_38_3055
Author(s) : Ordentlich A , Barak D , Kronman C , Benschop HP , De Jong LP , Ariel N , Barak R , Segall Y , Velan B , Shafferman A
Ref : Biochemistry , 38 :3055 , 1999
Abstract : The stereoselectivity of the phosphonylation reaction and the effects of adduct configuration on the aging process were examined for human acetylcholinesterase (HuAChE) and its selected active center mutants, using the four stereomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman). The reactivity of wild type HuAChE toward the PS-soman diastereomers was 4.0-7.5 x 10(4)-fold higher than that toward the PR-diastereomers. Aging of the PSCS-somanyl-HuAChE conjugate was also >1.6 x 10(4)-fold faster than that of the corresponding PRCS-somanyl adduct, as shown by both reactivation and electrospray mass spectrometry (ESI/MS) experiments. On the other hand, both processes exhibited very limited sensitivity to the chirality of the alkoxy group Calpha of either PS- or PR-diastereomers. These stereoselectivities presumably reflect the relative participation of the enzyme in stabilization of the Michaelis complexes and in dealkylation of the respective covalent conjugates, and therefore could be utilized for further probing of the HuAChE active center functional architecture. Reactivities of HuAChE enzymes carrying replacements at the acyl pocket (F295A, F297A, and F295L/F297V) indicate that stereoselectivity with respect to the soman phosphorus chirality depends on the structure of this binding subsite, but this stereoselectivity cannot be explained only by limitation in the capacity to accommodate the PR-diastereomers. In addition, these acyl pocket enzyme mutants display some (5-10-fold) preference for the PRCR-soman over the PRCS-stereomer, while reactivity of the hydrophobic pocket mutant enzyme W86F toward the PRCS-soman resembles that of the wild type HuAChE. Residue substitutions in the H-bond network (E202Q, E450A, Y133F, and Y133A) and the hydrophobic pocket (F338A, W86A, W86F, and Y337A) result in a limited stereoselectivity for the PSCS- over the PSCR-stereomer. Aging of the PS-somanyl conjugates with all the HuAChE mutant enzymes tested practically lacked stereoselectivity with respect to the Calpha of the alkoxy moiety. Thus, the inherent asymmetry of the active center does not seem to affect the rate-determining step of the dealkylation process, possibly because both the PSCS- and the PSCR-somanyl moieties yield the same carbocationic intermediate.
ESTHER : Ordentlich_1999_Biochemistry_38_3055
PubMedSearch : Ordentlich_1999_Biochemistry_38_3055
PubMedID: 10074358

Title : Contribution of Primary Sequence and Post-Translation Modification to the Pharmacokinetics of Human and Bovine Acetylcholinesterases -
Author(s) : Velan B , Kronman C , Chitlaru T , Mendelson I , Ordentlich A , Shafferman A
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. :291 , 1998
PubMedID:

Title : Compatibility of Structures Inferred from Mutagenesis and from X-Ray Crystallography for Various AChE Complexes -
Author(s) : Ariel N , Ordentlich A , Barak D , Bino T , Velan B , Shafferman A
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. :375 , 1998
PubMedID:

Title : The 'aromatic patch' of three proximal residues in the human acetylcholinesterase active centre allows for versatile interaction modes with inhibitors - Ariel_1998_Biochem.J_335_95
Author(s) : Ariel N , Ordentlich A , Barak D , Bino T , Velan B , Shafferman A
Ref : Biochemical Journal , 335 :95 , 1998
Abstract : The role of the functional architecture of the human acetylcholinesterase (HuAChE) active centre in accommodating the non-covalent inhibitors tacrine and huperzine A, or the carbamates pyridostigmine and physostigmine, was analysed using 16 mutants of residues lining the active-centre gorge. Despite the structural diversity of the ligands, certain common properties of the complexes could be observed: (a) replacement of aromatic residues Tyr133, Tyr337 and especially Trp86, resulted in pronounced changes in stability of all the complexes examined; (b) effects due to replacements of the five other aromatic residues along the active-centre gorge, such as the acyl pocket (Phe295, Phe297) or at the peripheral anionic site (Tyr124, Trp286, Tyr341) were relatively small; (c) effects due to substitution of the carboxylic residues in the gorge (Glu202, Glu450) were moderate. These results and molecular modelling indicate that the aromatic side chains of residues Trp86, Tyr133 and Tyr337 form together a continuous 'aromatic patch' lining the wall of the active-centre gorge, allowing for the accommodation of the different ligands via multiple modes of interaction. Studies with HuAChE mutants carrying replacements at positions 86, 133 and 337 indicate that the orientations of huperzine A and tacrine in the HuAChE complexes in solution are significantly different from those observed in X-ray structures of the corresponding complexes with Torpedo californica AChE (TcAChE). These discrepancies may be explained in terms of structural differences between the complexes of HuAChE and TcAChE or, more likely, by the enhanced flexibility of the AChE active-centre gorge in solution as compared with the crystalline state.
ESTHER : Ariel_1998_Biochem.J_335_95
PubMedSearch : Ariel_1998_Biochem.J_335_95
PubMedID: 9742217

Title : Crystal Structures of Aged Phosphorylated and Phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
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. :425 , 1998
PubMedID:

Title : Crystal structure of Aged phosphorylated and phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
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. :425 , 1998
PubMedID:
Gene_locus related to this paper: torca-ACHE

Title : Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels - Chitlaru_1998_Biochem.J_336_647
Author(s) : Chitlaru T , Kronman C , Zeevi M , Kam M , Harel A , Ordentlich A , Velan B , Shafferman A
Ref : Biochemical Journal , 336 :647 , 1998
Abstract : Sialylation of N-glycans associated with recombinant human acetylcholinesterase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their ability to sialylate overexpressed rHuAChE. High-resolution N-glycan structural analysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associated with rHuAChE produced in HEK-293 cells belong mainly to the complex-biantennary class and are only partly sialylated, with approx. 60% of the glycans being monosialylated. This partial sialylation characterizes rHuAChE produced by cells selected for high-level expression of the recombinant protein. In low-level producer lines, the enzyme exhibits a higher sialic acid content, suggesting that undersialylation of rHuAChE in high-level producer lines stems from a limited endogenous glycosyltransferase activity. To improve sialylation in HEK-293 cells, rat liver beta-galactoside alpha-2,6-sialyltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantly higher proportion of fully sialylated glycans as shown by sialic acid incorporation assays, direct measurement of sialic acid, and HPAEC glycan profiling. Genetically modified sialylated rHuAChE exhibited increased circulatory retention (the slow-phase half-life, t12beta, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increase in circulatory residence was observed when rHuAChE was subjected to extensive sialylation in vitro. The engineered HEK-293 cells in which the glycosylation machinery was modified might represent a valuable tool for the high level of expression of recombinant glycoproteins whose sialic acid content is important for their function or for pharmacokinetic behaviour.
ESTHER : Chitlaru_1998_Biochem.J_336_647
PubMedSearch : Chitlaru_1998_Biochem.J_336_647
PubMedID: 9841877

Title : Bovine Acetylcholinestrase Cloning, Expression and Characterization of the Recombinant Enzyme -
Author(s) : Mendelson I , Kronman C , Zeliger N , Seri T , Ordentlich A , Shafferman A , Velan B
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. :148 , 1998
PubMedID:

Title : Structural Modifications of the Omega Loop in Human Acetylcholinesterase -
Author(s) : Ariel N , Velan B , Barak D , Leitner M , Bino T , Ordentlich A , Shafferman A
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. :453 , 1998
PubMedID:

Title : Crystal Structures of Aged Phosphorylated and Phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
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. :454 , 1998
PubMedID:

Title : Functional characteristics of the oxyanion hole in human acetylcholinesterase - Ordentlich_1998_J.Biol.Chem_273_19509
Author(s) : Ordentlich A , Barak D , Kronman C , Ariel N , Segall Y , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 273 :19509 , 1998
Abstract : The contribution of the oxyanion hole to the functional architecture and to the hydrolytic efficiency of human acetylcholinesterase (HuAChE) was investigated through single replacements of its elements, residues Gly-121, Gly-122 and the adjacent residue Gly-120, by alanine. All three substitutions resulted in about 100-fold decrease of the bimolecular rate constants for hydrolysis of acetylthiocholine; however, whereas replacements of Gly-120 and Gly-121 affected only the turnover number, mutation of residue Gly-122 had an effect also on the Michaelis constant. The differential behavior of the G121A and G122A enzymes was manifested also toward the transition state analog m-(N,N, N-trimethylammonio)trifluoroacetophenone (TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward TMTFA was 2000-11, 000-fold lower than that of the wild type HuAChE; however, the G121A enzyme exhibited a rapid inhibition pattern, as opposed to the slow binding kinetics shown by the G122A enzyme. For both phosphates (diethyl phosphorofluoridate, diisopropyl phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman), the decrease in inhibitory activity toward the G121A enzyme was very substantial (2000-6700-fold), irrespective of size of the alkoxy substituents on the phosphorus atom. On the other hand, for the G122A HuAChE the relative decline in reactivity toward phosphonates (500-460-fold) differed from that toward the phosphates (12-95-fold). Although formation of Michaelis complexes with substrates does not seem to involve significant interaction with the oxyanion hole, interactions with this motif are a major stabilizing element in accommodation of covalent inhibitors like organophosphates or carbamates. These observations and molecular modeling suggest that replacements of residues Gly-120 or Gly-121 by alanine alter the structure of the oxyanion hole motif, abolishing the H-bonding capacity of residue at position 121. These mutations weaken the interaction between HuAChE and the various ligands by 2.7-5.0 kcal/mol. In contrast, variations in reactivity due to replacement of residue Gly 122 seem to result from steric hindrance at the active center acyl pocket
ESTHER : Ordentlich_1998_J.Biol.Chem_273_19509
PubMedSearch : Ordentlich_1998_J.Biol.Chem_273_19509
PubMedID: 9677373

Title : Contribution of the Active Center Functional Architecture to AChE Reactivity Toward Substrates and Inhibitors -
Author(s) : Shafferman A , Ordentlich A , Barak D , Kronman C , Ariel N , Velan B
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. :203 , 1998
PubMedID:

Title : Does Electrostatic Attraction or Steering by Charged Residues within the Gorge Contribute to the Reactivity of AChE? -
Author(s) : Ordentlich A , Barak D , Stein D , Berman D , Kronman C , Ariel N , Velan B , Shafferman A
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. :234 , 1998
PubMedID:

Title : The Aromatic Moiety at Position-86 of HuAChE Accelerate the Aging of Phosphonyl-AChE Conjugates through Cation- Interactions -
Author(s) : Barak D , Ordentlich A , Stein D , Segall Y , Velan B , Benschop HP , De Jong LP , Shafferman A
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. :246 , 1998
PubMedID:

Title : ESMS as a Unique Tool for the Molecular Monitoring of Reactions between HuAChE and Various OP-Agents -
Author(s) : Ordentlich A , Barak R , Barak D , Fischer M , Benschop HP , De Jong LP , Segall Y , Velan B , Shafferman A
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. :249 , 1998
PubMedID:

Title : Aging of somanyl-acetylcholinesterase adducts: facts and models [letter] -
Author(s) : Shafferman A , Ordentlich A , Barak D , Stein D , Ariel N , Velan B
Ref : Biochemical Journal , 324 :996 , 1997
PubMedID: 9235881

Title : Direct determination of the chemical composition of acetylcholinesterase phosphonylation products utilizing electrospray- ionization mass spectrometry - Barak_1997_FEBS.Lett_407_347
Author(s) : Barak R , Ordentlich A , Barak D , Fischer M , Benschop HP , De Jong LP , Segall Y , Velan B , Shafferman A
Ref : FEBS Letters , 407 :347 , 1997
Abstract : While non-reactivability of cholinesterases from their phosphyl conjugates (aging) is attributed to an unimolecular process involving loss of alkyl group from the phosphyl moiety, no conclusive evidence is available that this is the only reaction path and involvement of other post-inhibitory processes cannot be ruled out. To address this issue, molecular masses of the bacterially expressed recombinant human acetylcholinesterase and of its conjugates with a homologous series of alkyl methylphosphonofluoridates, were measured by electrospray-ionization mass spectrometry (ESI-MS). The measured mass of the free enzyme was 64,700 Da (calculated 64,695 Da) and those of the methylphosphono-HuAChE adducts, bearing isopropyl, isobutyl, 1,2-dimethylpropyl and 1,2,2-trimethylpropyl substituents, were 64,820, 64,840, 64,852 and 64,860 Da, respectively. These values reflect both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of these adducts change with time to yield a common product with molecular mass of 64,780 Da which is consistent with dealkylation of the phosphonyl moieties. Furthermore, in the case of 1,2-dimethylpropyl methylphosphono-HuAChE, the change in the molecular mass and the kinetics of non-reactivability appear to occur in parallel indicating that dealkylation is indeed the predominant molecular transformation leading to 'aging' of phosphonyl-AChE adducts.
ESTHER : Barak_1997_FEBS.Lett_407_347
PubMedSearch : Barak_1997_FEBS.Lett_407_347
PubMedID: 9175882

Title : Structural modifications of the omega loop in human acetylcholinesterase - Velan_1996_FEBS.Lett_395_22
Author(s) : Velan B , Barak D , Ariel N , Leitner M , Bino T , Ordentlich A , Shafferman A
Ref : FEBS Letters , 395 :22 , 1996
Abstract : Conformational mobility of the surface omega loop (Cys-69-Cys-96) in human acetylcholinesterase (HuAChE) was recently implicated in substrate accessibility to the active center and in the mechanism of allosteric modulation of enzymatic activity. We therefore generated and kinetically evaluated the following modifications or replacements in HuAChE: (a) residues at the loop ends, (b) residues involved in putative hydrogen-bond interactions within the loop and between the loop and the protein core, (c) ChEs conserved proline residues within the loop and (d) a deletion of a conserved segment of 5 residues. All the residue replacements, including those of the prolines, had either limited or no effect on enzyme reactivity. These results suggest that unlike the case of lipase, the omega loop in the HuAChE is not involved in large lid-like displacements. In cases where modifications of the loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp-86 supporting the proposed coupling between the structure of the omega loop and the positioning of the Trp-86 indole moiety, in catalytic activity and in allosterism.
ESTHER : Velan_1996_FEBS.Lett_395_22
PubMedSearch : Velan_1996_FEBS.Lett_395_22
PubMedID: 8849682

Title : Aging of phosphylated human acetylcholinesterase: catalytic processes mediated by aromatic and polar residues of the active centre - Shafferman_1996_Biochem.J_318_833
Author(s) : Shafferman A , Ordentlich A , Barak D , Stein D , Ariel N , Velan B
Ref : Biochemical Journal , 318 :833 , 1996
Abstract : We have examined the effects of 11 substitutions of active centre gorge residues of human acetylcholinesterase (HuAChE) on the rates of phosphonylation by 1,2,2-trimethylpropyl methyl-phosphonofluoridate (soman) and the aging of the resulting conjugates. The rates of phosphonylation were reduced to as little as one-seventieth, mainly in mutants of the hydrogen-bond network (Glu-202, Glu-450, Tyr-133). These recombinant enzymes as well as the F338A, W86A, W86F and D74N mutant HuAChEs varied in their resistance to aging (15-3300-fold relative to the wild type). The most dramatic resistance to aging was observed for the phosphonyl conjugate of the mutant W86A enzyme (1850-3300-fold relative to the wild type). It is proposed that Trp-86 contributes to the aging process by stabilizing the evolving carbonium ion on the 1,2,2-trimethylpropyl moiety, via charge-pi interaction. The rate-enhancing effect of Trp-86 provides a rationale for the unique facility of aging in soman-inhibited cholinesterases, compared with the corresponding conjugates in other serine hydrolases. Replacements of Glu-202 by aspartic acid, glutamine or alanine residues resulted in a similar (1/130-1/300) decrease of the rates of aging. A comparable decrease was also observed for the conjugate of the F338A mutant. These results, and the similar pH dependence of aging rates for the wild-type and E202Q and F338A mutant HuAChEs, indicate that Glu-202 is not involved in proton transfer to the phosphonyl moiety. On the basis of these findings and of molecular modelling we suggest that Glu-202 and Phe-338 contribute to the aging process by stabilizing the imidazolium of the catalytic triad His-447 via charge-charge and charge-pi interactions respectively, thereby facilitating an oxonium formation on the phosphonyl moiety.
ESTHER : Shafferman_1996_Biochem.J_318_833
PubMedSearch : Shafferman_1996_Biochem.J_318_833
PubMedID: 8836126

Title : Interactions of oxime reactivators with diethylphosphoryl adducts of human acetylcholinesterase and its mutant derivatives - Grosfeld_1996_Mol.Pharmacol_50_639
Author(s) : Grosfeld H , Barak D , Ordentlich A , Velan B , Shafferman A
Ref : Molecular Pharmacology , 50 :639 , 1996
Abstract : Diethylphosphoryl conjugates of human acetylcholinesterase (AChE) and selected mutants, carrying amino acid replacements at the active center and at the peripheral anionic site, were subjected to reactivation with the monopyridinium oxime 2-hydroxy-iminomethyl-1-methylpyridinium chloride and the bispyridinium oximes 1,3-bis(4'-hydroxyiminomethyl-1'-pyridinium),propane dibromide (TMB-4(Trimedoxime)) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"-pyridinium)-2 - oxapropane dichloride (HI-6). The kinetic profiles for all of the reactivation reactions indicate single populations of reactivatable species. Replacement of Trp86, the anionic subsite in the active center, lowered the affinity of the free enzyme toward all three reactivators, but in the corresponding diethylphosphoryl conjugate, only affinity toward TMB-4(Trimedoxime) was affected. Replacement of other constituents of the hydrophobic subsite (Tyr337, Phe338) had no major effect on either affinity to the free enzymes or rates of reactivation. Substitution of residues of the acyl pocket (Phe295, Phe297) lowered the affinities toward reactivators except for the 20-fold increase in affinity of F295A toward HI-6. Replacement of the acidic residues in the active center (Glu202, Glu450) affected mainly the rates of nucleophilic displacement of the phosphoryl moiety. The effect of substituting residues constituting the peripheral anionic site at the rim of the active site gorge (Tyr72, Asp74, Trp286) was particularly puzzling because for 2-hydroxy-iminomethyl-1-methylpyridinium chloride and HI-6, mainly the nucleophilic reaction rate constants were affected, whereas for TMB-4(Trimedoxime), the affinities of the phosphorylated enzymes were significantly reduced. The fact that perturbations of the functional architecture of HuAChE active center can account for only some of the observed effects on the reactivation rates suggests that the binding modes of oxime to the phosphorylated and nonphosphorylated enzymes are considerably different and/or that interactions of the reactivators with the phosphoryl moieties play a dominant role in the reactivation process.
ESTHER : Grosfeld_1996_Mol.Pharmacol_50_639
PubMedSearch : Grosfeld_1996_Mol.Pharmacol_50_639
PubMedID: 8794905

Title : The architecture of human acetylcholinesterase active center probed by interactions with selected organophosphate inhibitors - Ordentlich_1996_J.Biol.Chem_271_11953
Author(s) : Ordentlich A , Barak D , Kronman C , Ariel N , Segall Y , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 271 :11953 , 1996
Abstract : The role of the functional architecture of human acetylcholinesterase (HuAChE) active center in facilitating reactions with organophosphorus inhibitors was examined by a combination of site-directed mutagenesis and kinetic studies of phosphorylation with organophosphates differing in size of their alkoxy substituents and in the nature of the leaving group. Replacements of residues Phe-295 and Phe-297, constituting the HuAChE acyl pocket, increase up to 80-fold the reactivity of the enzymes toward diisopropyl phosphorofluoridate, diethyl phosphorofluoridate, and p-nitrophenyl diethyl phosphate (paraoxon), indicating the role of this subsite in accommodating the phosphate alkoxy substituent. On the other hand, a decrease of up to 160-fold in reactivity was observed for enzymes carrying replacements of residues Tyr-133, Glu-202, and Glu-450, which are constituents of the hydrogen bond network in the HuAChE active center, which maintains its unique functional architecture. Replacement of residues Trp-86, Tyr-337, and Phe-338 in the alkoxy pocket affected reactivity toward diisopropyl phosphorofluoridate and paraoxon, but to a lesser extent that toward diethyl phosphorofluoridate, indicating that both the alkoxy substituent and the p-nitrophenoxy leaving group interact with this subsite. In all cases the effects on reactivity toward organophosphates, demonstrated in up to 10,000-fold differences in the values of bimolecular rate constants, were mainly a result of altered affinity of the HuAChE mutants, while the apparent first order rate constants of phosphorylation varied within a narrow range. This finding indicates that the main role of the functional architecture of HuAChE active center in phosphorylation is to facilitate the formation of enzyme-inhibitor Michaelis complexes and that this affinity, rather than the nucleophilic activity of the enzyme catalytic machinery, is a major determinant of HuAChE reactivity toward organophosphates.
ESTHER : Ordentlich_1996_J.Biol.Chem_271_11953
PubMedSearch : Ordentlich_1996_J.Biol.Chem_271_11953
PubMedID: 8662593

Title : Molecular Aspects of Catalysis and of Allosteric Regulation of Aceytlcholinesterases -
Author(s) : Shafferman A , Ordentlich A , Barak D , Kronman C , Ariel N , Leitner M , Segall Y , Bromberg A , Reuveny S , Marcus D , Bino T , Lazar A , Cohen S , Velan B
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. :189 , 1995
PubMedID:

Title : Contribution of aromatic moieties of tyrosine 133 and of the anionic subsite tryptophan 86 to catalytic efficiency and allosteric modulation of acetylcholinesterase - Ordentlich_1995_J.Biol.Chem_270_2082
Author(s) : Ordentlich A , Barak D , Kronman C , Ariel N , Segall Y , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 270 :2082 , 1995
Abstract : Substitution of Trp-86, in the active center of human acetylcholinesterase (HuAChE), by aliphatic but not by aromatic residues resulted in a several thousandfold decrease in reactivity toward charged substrate and inhibitors but only a severalfold decrease for noncharged substrate and inhibitors. The W86A and W86E HuAChE enzymes exhibit at least a 100-fold increase in the Michaelis-Menten constant or 100-10,000-fold increase in inhibition constants toward various charged inhibitors, as compared to W86F HuAChE or the wild type enzyme. On the other hand, replacement of Glu-202, the only acidic residue proximal to the catalytic site, by glutamine resulted in a nonselective decrease in reactivity toward charged and noncharged substrates or inhibitors. Thus, the quaternary nitrogen groups of substrates and other active center ligands, are stabilized by cation-aromatic interaction with Trp-86 rather than by ionic interactions, while noncharged ligands appear to bind to distinct site(s) in HuAChE. Analysis of the Y133F and Y133A HuAChE mutated enzymes suggests that the highly conserved Tyr-133 plays a dual role in the active center: (a) its hydroxyl appears to maintain the functional orientation of Glu-202 by hydrogen bonding and (b) its aromatic moiety maintains the functional orientation of the anionic subsite Trp-86. In the absence of aromatic interactions between Tyr-133 and Trp-86, the tryptophan acquires a conformation that obstructs the active site leading, in the Y133A enzyme, to several hundredfold decrease in rates of catalysis, phosphorylation, or in affinity to reversible active site inhibitors. It is proposed that allosteric modulation of acetylcholinesterase activity, induced by binding to the peripheral anionic sites, proceeds through such conformational change of Trp-86 from a functional anionic subsite state to one that restricts access of substrates to the active center.
ESTHER : Ordentlich_1995_J.Biol.Chem_270_2082
PubMedSearch : Ordentlich_1995_J.Biol.Chem_270_2082
PubMedID: 7836436

Title : Compilation of Evaluated Mutants of Cholinesterases -
Author(s) : Shafferman A , Kronman C , Ordentlich 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. :481 , 1995
PubMedID:

Title : Involvement of oligomerization, N-glycosylation and sialylation in the clearance of cholinesterases from the circulation - Kronman_1995_Biochem.J_311_959
Author(s) : Kronman C , Velan B , Marcus D , Ordentlich A , Reuveny S , Shafferman A
Ref : Biochemical Journal , 311 :959 , 1995
Abstract : The possible role of post-translational modifications such as subunit oligomerization, protein glycosylation and oligosaccharide processing on the circulatory life-time of proteins was studied using recombinant human acetylcholinesterase (rHuAChE). Different preparations of rHuAChE containing various amounts of tetramers, dimers and monomers are cleared at similar rates from the circulation, suggesting that oligomerization does not play an important role in determining the rate of clearance. An engineered rHuAChE mutant containing only one N-glycosylation site was cleared from the circulation more rapidly than the wild-type triglycosylated enzyme. On the other hand, hyperglycosylated mutants containing either four or five occupied N-glycosylation sites, analagous to those present on the slowly cleared fetal bovine serum acetylcholinesterase (FBS-AChE), were also cleared more rapidly from the bloodstream than the wild-type species. Furthermore, the two different tetraglycosylated mutants were cleared at different rates while the pentaglycosylated mutant exhibited the most rapid clearance profile. These results imply that though the number of N-glycosylation sites plays a role in the circulatory life-time of the enzyme, the number of N-glycan units in itself does not determine the rate of clearance. When saturating amounts of asialofetuin were administered together with rHuAChE, the circulatory half-life of the enzyme was dramatically increased (from 80 min to 19 h) and was found to be similar to that displayed by plasma-derived cholinesterases while desialylation of these enzymes caused a sharp decrease in the circulatory half-life to approximately 3-5 min. Determination of the average number of sialic acid residues per enzyme subunit of the five different N-glycosylation species generated, revealed that the rate of clearance is not a function of the absolute number of appended sialic acid moieties but rather of the number of unoccupied sialic acid attachment sites per enzyme molecule. Specifically, we demonstrate an inverse-linear relationship between the number of vacant sialic acid attachment sites and the values of the enzyme residence time within the bloodstream.
ESTHER : Kronman_1995_Biochem.J_311_959
PubMedSearch : Kronman_1995_Biochem.J_311_959
PubMedID: 7487957

Title : Electrostatic Attraction by Surface Charge does not Contribute to the Catalytic Efficiency of Acetylcholinesterase -
Author(s) : Barak D , Ordentlich A , Kronman C , Ber R , Bino T , Ariel N , Osman R , 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. :223 , 1995
PubMedID:

Title : Amino Acids Determining Specificity to OP-Agents and Facilitating the Aging Process in Human Acetylcholinesterase -
Author(s) : Ordentlich A , Kronman C , Stein D , Ariel N , Reuveny S , Marcus D , Segall Y , Barak 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. :221 , 1995
PubMedID:

Title : Post-Translation Processing of Acetylcholinesterase -
Author(s) : Velan B , Kronman C , Ordentlich A , Flashner Y , Ber R
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. :269 , 1995
PubMedID:

Title : Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite - Barak_1995_Biochemistry_34_15444
Author(s) : Barak D , Ordentlich A , Bromberg A , Kronman C , Marcus D , Lazar A , Ariel N , Velan B , Shafferman A
Ref : Biochemistry , 34 :15444 , 1995
Abstract : Replacement of residues Asp74, Trp286, and Tyr72, which are constituents of the peripheral anionic site (PAS) of human acetylcholinesterase (HuAChE), affected similarly both the binding and the inhibition constants of the PAS-specific ligand propidium, demonstrating that changes in the inhibitory activity are a direct consequence of altered binding to the PAS. In contrast, the active center HuAChE mutants W86A and Y133A show respective 350- and 25-fold increased resistance to inhibition by propidium but no change in binding affinities, demonstrating that the allosteric mechanism of PAS-mediated inhibition involves a conformational change of these Trp86 and Tyr133 residues rather than physical obstruction of substrate access by the inhibitor itself. These findings support the recent proposal that the allosteric mechanism operates via transition between active and nonactive conformations of the anionic subsite Trp86 and that replacement of Tyr133 by alanine may stabilize a nonactive Trp86 conformation that occludes the active center [Ordentlich et al. (1995) J. Biol. Chem. 270, 2082]. In further support of this mechanism and the role of Tyr133, we find that (a) the dissociation constants (Kd) for the noncovalent complexes of the irreversible inhibitors diisopropyl phosphorofluoridate or paraoxon with Y133A HuAChE are increased 20-500-fold, relative to either wild-type enzyme or its Y133F or W86A mutants; and (b) access of substrates such as 3,3-dimethylbutyl thioacetate is restored by removal of Trp86 from the Y133A enzyme (i.e., the W86A/Y133A mutant). We suggest that the conformational transition of Trp86 is coupled to the motions of the cysteine loop (Cys69-Cys96) of HuAChE and is inherent to the dynamics of the native enzyme.
ESTHER : Barak_1995_Biochemistry_34_15444
PubMedSearch : Barak_1995_Biochemistry_34_15444
PubMedID: 7492545

Title : Acetylcholinesterase peripheral anionic site degeneracy conferred by amino acid arrays sharing a common core - Barak_1994_J.Biol.Chem_269_6296
Author(s) : Barak D , Kronman C , Ordentlich A , Ariel N , Bromberg A , Marcus D , Lazar A , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 269 :6296 , 1994
Abstract : Several of the residues constituting the peripheral anionic site (PAS) in human acetylcholinesterase (HuAChE) were identified by a combination of kinetic studies with 19 single and multiple HuAChE mutants, fluorescence binding studies with the Trp-286 mutant, and by molecular modeling. Mutants were analyzed with three structurally distinct positively charged PAS ligands, propidium, decamethonium, and di(p-allyl-N-dimethylaminophenyl)pentane-3-one (BW284C51), as well as with selective active center inhibitors, hexamethonium and edrophonium. Single mutations of residues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341 resulted in up to 10-fold increase in inhibition constants for PAS ligands, whereas for multiple mutants up to 400-fold increase was observed. The 6th PAS element residue Asp-74 is unique in its ability to affect conformation of both the active site and the PAS (Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992) EMBO J. 11, 3561-3568) as demonstrated by the several hundred-fold increase in Ki for D74N inhibition by the bisquaternary ligands decamethonium and BW284C51. Based on these studies, singular molecular models for the various HuAChE inhibitor complexes were defined. Yet, for the decamethonium complex two distinct conformations were generated, accommodating the quaternary ammonium group by interactions with either Trp-286 or with Tyr-341. We propose that the PAS consists of a number of binding sites, close to the entrance of the active site gorge, sharing residues Asp-74 and Trp-286 as a common core. Binding of ligands to these residues may be the key to the allosteric modulation of HuAChE catalytic activity. This functional degeneracy is a result of the ability of the Trp-286 indole moiety to interact either via stacking, aromatic-aromatic, or via pi-cation attractions and the involvement of the carboxylate of Asp-74 in charge-charge or H-bond interactions.
ESTHER : Barak_1994_J.Biol.Chem_269_6296
PubMedSearch : Barak_1994_J.Biol.Chem_269_6296
PubMedID: 8119978

Title : The back door hypothesis for product clearance in acetylcholinesterase challenged by site-directed mutagenesis - Kronman_1994_J.Biol.Chem_269_27819
Author(s) : Kronman C , Ordentlich A , Barak D , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 269 :27819 , 1994
Abstract : The active site of acetylcholinesterase is near the bottom of a long and narrow gorge. The dimensions of the gorge and the strong electrostatic field generated by the enzyme appear inconsistent with the enzyme's high turnover rate. Consequently, a "back door" mechanism involving movement of the reaction products through a transient opening near the active center was recently suggested. We investigated this hypothesis in human acetylcholinesterase by testing mutants at key residues (Glu-84, Trp-86, Asp-131, and Val-132) located near or along the putative back door channel. The turnover rates of all mutants tested, and in particular of V132K, where the channel is expected to be sealed by salt bridge Lys-132-Glu-452, are similar to that of the wild type enzyme. This indicates that the proposed back door is not a route for product clearance from the active site gorge of acetylcholinesterase and is probably of no functional relevance to its catalytic activity.
ESTHER : Kronman_1994_J.Biol.Chem_269_27819
PubMedSearch : Kronman_1994_J.Biol.Chem_269_27819
PubMedID: 7961709

Title : Electrostatic attraction by surface charge does not contribute to the catalytic efficiency of acetylcholinesterase - Shafferman_1994_EMBO.J_13_3448
Author(s) : Shafferman A , Ordentlich A , Barak D , Kronman C , Ber R , Bino T , Ariel N , Osman R , Velan B
Ref : EMBO Journal , 13 :3448 , 1994
Abstract : Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.
ESTHER : Shafferman_1994_EMBO.J_13_3448
PubMedSearch : Shafferman_1994_EMBO.J_13_3448
PubMedID: 8062821

Title : Engineering resistance to 'aging' of phosphylated human acetylcholinesterase. Role of hydrogen bond network in the active center - Ordentlich_1993_FEBS.Lett_334_215
Author(s) : Ordentlich A , Kronman C , Barak D , Stein D , Ariel N , Marcus D , Velan B , Shafferman A
Ref : FEBS Letters , 334 :215 , 1993
Abstract : Recombinant human acetylcholinesterase (HuAChE) and selected mutants (E202Q, Y337A, E450A) were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation with organophosphorus (OP) inhibitors and subsequent aging of the OP-conjugates. Amino acid E450, unlike residues E202 and Y337, is not within interaction distance from the active center. Yet, the bimolecular rates of catalysis and phosphylation are 30-100 fold lower for both E450A and E202Q compared to Y337A or the wild type and in both mutants the resulting OP-conjugates show striking resistance to aging. It is proposed that a hydrogen bond network, that maintains the functional architecture of the active center, involving water molecules and residues E202 and E450, is responsible for the observed behaviour.
ESTHER : Ordentlich_1993_FEBS.Lett_334_215
PubMedSearch : Ordentlich_1993_FEBS.Lett_334_215
PubMedID: 8224249

Title : Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Identification of residues constituting the anionic site, the hydrophobic site, and the acyl pocket - Ordentlich_1993_J.Biol.Chem_268_17083
Author(s) : Ordentlich A , Barak D , Kronman C , Flashner Y , Leitner M , Segall Y , Ariel N , Cohen S , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 268 :17083 , 1993
Abstract : Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.
ESTHER : Ordentlich_1993_J.Biol.Chem_268_17083
PubMedSearch : Ordentlich_1993_J.Biol.Chem_268_17083
PubMedID: 8349597
Gene_locus related to this paper: human-ACHE , human-BCHE

Title : N-glycosylation of human acetylcholinesterase: effects on activity, stability and biosynthesis - Velan_1993_Biochem.J_296_649
Author(s) : Velan B , Kronman C , Ordentlich A , Flashner Y , Leitner M , Cohen S , Shafferman A
Ref : Biochemical Journal , 296 :649 , 1993
Abstract : The role of N-glycosylation in the function of human acetylcholinesterase (HuAChE) was examined by site-directed mutagenesis (Asn to Gln substitution) of the three potential N-glycosylation sites Asn-265, Asn-350 and Asn-464. Analysis of HuAChE mutants, defective in a single or multiple N-glycosylation sites, by expression in transiently or stably transfected human embryonal 293 kidney cells suggests the following. (a) All three AChE glycosylation signals are utilized, but not all the secreted molecules are fully glycosylated. (b) Glycosylation at all sites is important for effective biosynthesis and secretion; extracellular AChE levels in mutants defective in one, two or all three sites amounted to 20-30%, 2-4% and about 0.5% of wild-type level respectively. (c) Some glycosylation mutants display impaired stability, as reflected by increased susceptibility to heat inactivation; substitution of Asn-464 has the most pronounced effect on thermostability. (d) Abrogation of N-glycosylation has no detectable effect on the enzyme activity of HuAChE; all glycosylation mutants, including the triple mutant, hydrolyse acetylthiocholine efficiently, displaying Km, kcat. and kcat./Km values similar to those of the wild-type enzyme. (e) In most mutants, inhibition profiles with edrophonium and bisquaternary ammonium ligands are identical with those of wild-type enzyme; the Asn-350 mutants, however, exhibit a slight decrease in their affinity towards these ligands. (f) Elimination of oligosaccharide side chains has no detectable effect on the surface-related 'peripheral-site' functions; like the wild-type enzyme, all mutants were inhibited by propidium and by increased concentrations of acetylthiocholine.
ESTHER : Velan_1993_Biochem.J_296_649
PubMedSearch : Velan_1993_Biochem.J_296_649
PubMedID: 8280063

Title : Recombinant human acetylcholinesterase - Enzyme engineering -
Author(s) : Shafferman A , Velan B , Barak D , Kronman C , Ordentlich A , Flashner Y , Leitner M , Segal Y , Grosfeld H , Stein D , Ariel N
Ref : Medical Defense Bioscience Review , 3 :1097 , 1993
PubMedID:

Title : Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding - Shafferman_1992_J.Biol.Chem_267_17640
Author(s) : Shafferman A , Kronman C , Flashner Y , Leitner M , Grosfeld H , Ordentlich A , Gozes Y , Cohen S , Ariel N , Barak D , Harel M , Silman I , Sussman JL , Velan B
Ref : Journal of Biological Chemistry , 267 :17640 , 1992
Abstract : Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.
ESTHER : Shafferman_1992_J.Biol.Chem_267_17640
PubMedSearch : Shafferman_1992_J.Biol.Chem_267_17640
PubMedID: 1517212

Title : Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center - Shafferman_1992_EMBO.J_11_3561
Author(s) : Shafferman A , Velan B , Ordentlich A , Kronman C , Grosfeld H , Leitner M , Flashner Y , Cohen S , Barak D , Ariel N
Ref : EMBO Journal , 11 :3561 , 1992
Abstract : Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.
ESTHER : Shafferman_1992_EMBO.J_11_3561
PubMedSearch : Shafferman_1992_EMBO.J_11_3561
PubMedID: 1396557