Rosenberry TL

General

Full name : Rosenberry Terrone L

First name : Terrone

Mail : Department of Neuroscience\; Mayo Clinic Florida\; 4500 San Pablo Road, Birdsall 213,Jacksonville\; FL 32224

Zip Code :

City : Jacksonville

Country : USA

Email : rosenberry@mayo.edu

Phone : +19049537375

Fax : 1-904-953-7370

Website : \/\/\/www.mayo.edu\/research\/faculty\/rosenberry-terrone-l-ph-d\/BIO-00027368

Directory :

References (110)

Title : Solvent Deuterium Oxide Isotope Effects on the Reactions of Organophosphorylated Acetylcholinesterase - Rosenberry_2020_Molecules_25_
Author(s) : Rosenberry TL
Ref : Molecules , 25 : , 2020
Abstract : Organophosphates (OPs) are esters of substituted phosphates, phosphonates or phosphoramidates that react with acetylcholinesterase (AChE) by initially transferring the organophosphityl group to a serine residue in the enzyme active site, concomitant with loss of an alcohol or halide leaving group. With substituted phosphates, this transfer is followed by relatively slow hydrolysis of the organophosphoryl AChE, or dephosphorylation, that is often accompanied by an aging reaction that renders the enzyme irreversibly inactivated. Aging is a dealkylation that converts the phosphate triester to a diester. OPs are very effective AChE inhibitors and have been developed as insecticides and chemical warfare agents. We examined three reactions of two organophosphoryl AChEs, dimethyl- and diethylphosphorylated AChE, by comparing rate constants and solvent deuterium oxide isotope effects for hydrolysis, aging and oxime reactivation with pralidoxime (2-PAM). Our study was motivated (1) by a published x-ray crystal structure of diethylphosphorylated AChE, which showed severe distortion of the active site that was restored by the binding of pralidoxime, and (2) by published isotope effects for decarbamoylation that decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE. We previously reconciled these results by proposing a shift in the rate-limiting step from proton transfer for the small carbamoyl group to a likely conformational change in the distorted active site of the large carbamoyl enzyme. This proposal was tested but was not supported in this report. The smaller dimethylphosphoryl AChE and the larger diethylphosphoryl AChE gave similar isotope effects for both oxime reactivation and hydrolysis, and the isotope effect values of about two indicated that proton transfer was rate limiting for both reactions.
ESTHER : Rosenberry_2020_Molecules_25_
PubMedSearch : Rosenberry_2020_Molecules_25_
PubMedID: 32992925

Title : A Second Look at the Crystal Structures of Drosophila melanogaster Acetylcholinesterase in Complex with Tacrine Derivatives Provides Insights Concerning Catalytic Intermediates and the Design of Specific Insecticides - Nachon_2020_Molecules_25_1198
Author(s) : Nachon F , Rosenberry TL , Silman I , Sussman JL
Ref : Molecules , 25 :1198 , 2020
Abstract : Over recent decades, crystallographic software for data processing and structure refinement has improved dramatically, resulting in more accurate and detailed crystal structures. It is, therefore, sometimes valuable to have a second look at 'old' diffraction data, especially when earlier interpretation of the electron density maps was rather difficult. Here, we present updated crystal structures of Drosophila melanogaster acetylcholinesterase (DmAChE) originally published in [Harel et al., Prot Sci (2000) 9:1063-1072], which reveal features previously unnoticed. Thus, previously unmodeled density in the native active site can be interpreted as stable acetylation of the catalytic serine. Similarly, a strong density in the DmAChE/ZA complex originally attributed to a sulfate ion is better interpreted as a small molecule that is covalently bound. This small molecule can be modeled as either a propionate or a glycinate. The complex is reminiscent of the carboxylate butyrylcholinesterase complexes observed in crystal structures of human butyrylcholinesterases from various sources, and demonstrates the remarkable ability of cholinesterases to stabilize covalent complexes with carboxylates. A very strong peak of density (10 sigma) at covalent distance from the C beta of the catalytic serine is present in the DmAChE/ZAI complex. This can be undoubtedly attributed to an iodine atom, suggesting an unanticipated iodo/hydroxyl exchange between Ser238 and the inhibitor, possibly driven by the intense X-ray irradiation. Finally, the binding of tacrine-derived inhibitors, such as ZA (1DX4) or the iodinated analog, ZAI (1QON) results in the appearance of an open channel that connects the base of the active-site gorge to the solvent. This channel, which arises due to the absence of the conserved tyrosine present in vertebrate cholinesterases, could be exploited to design inhibitors specific to insect cholinesterases. The present study demonstrates that updated processing of older diffraction images, and the re-refinement of older diffraction data, can produce valuable information that could not be detected in the original analysis, and strongly supports the preservation of the diffraction images in public data banks.
ESTHER : Nachon_2020_Molecules_25_1198
PubMedSearch : Nachon_2020_Molecules_25_1198
PubMedID: 32155891
Gene_locus related to this paper: drome-ACHE

Title : Rate-limiting step in the decarbamoylation of acetylcholinesterases with large carbamoyl groups - Rosenberry_2019_Chem.Biol.Interact_13ChEPon_
Author(s) : Rosenberry TL , Cheung J
Ref : Chemico-Biological Interactions , : , 2019
Abstract : Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. In this report, we review recent data showing that decarbamoylation rate constants are independent of the ester leaving group for a series of carbamic acid esters with the same carbamoyl group and that decarbamoylation rate constants decreased by 800-fold when the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-diethyl-. We also review data showing that solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site in N,N-diethylcarbamoyl AChE. The nature of such a conformational change is suggested from X-ray crystal structures of AChE phosphorylated by paraoxon.
ESTHER : Rosenberry_2019_Chem.Biol.Interact_13ChEPon_
PubMedSearch : Rosenberry_2019_Chem.Biol.Interact_13ChEPon_
PubMedID: 31175846

Title : Decarbamoylation of acetylcholinesterases is markedly slowed as carbamoyl groups increase in size - Venkatasubban_2018_Arch.Biochem.Biophys_655_67
Author(s) : Venkatasubban KS , Johnson JL , Thomas JL , Fauq A , Cusack B , Rosenberry TL
Ref : Archives of Biochemistry & Biophysics , 655 :67 , 2018
Abstract : Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. We show here, in contrast to a previous report, that decarbamoylation rate constants are independent of the leaving group for a series of carbamates with the same carbamoyl group. When the alkyl substituents on the carbamoyl group increased in size from N-monomethyl- to N,N-dimethyl-, N-ethyl-N-methyl-, or N,N-diethyl-, the decarbamoylation rate constants decreased by 4-, 70-, and 800-fold, respectively. We suggest that this relationship arises as a result of active site distortion, particularly in the acyl pocket of the active site. Furthermore, solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site.
ESTHER : Venkatasubban_2018_Arch.Biochem.Biophys_655_67
PubMedSearch : Venkatasubban_2018_Arch.Biochem.Biophys_655_67
PubMedID: 30098983

Title : Comparison of the Binding of Reversible Inhibitors to Human Butyrylcholinesterase and Acetylcholinesterase: A Crystallographic, Kinetic and Calorimetric Study - Rosenberry_2017_Molecules_22_
Author(s) : Rosenberry TL , Brazzolotto X , Macdonald IR , Wandhammer M , Trovaslet-Leroy M , Darvesh S , Nachon F
Ref : Molecules , 22 : , 2017
Abstract : Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) hydrolyze the neurotransmitter acetylcholine and, thereby, function as coregulators of cholinergic neurotransmission. Although closely related, these enzymes display very different substrate specificities that only partially overlap. This disparity is largely due to differences in the number of aromatic residues lining the active site gorge, which leads to large differences in the shape of the gorge and potentially to distinct interactions with an individual ligand. Considerable structural information is available for the binding of a wide diversity of ligands to AChE. In contrast, structural data on the binding of reversible ligands to BChE are lacking. In a recent effort, an inhibitor competition approach was used to probe the overlap of ligand binding sites in BChE. Here, we extend this study by solving the crystal structures of human BChE in complex with five reversible ligands, namely, decamethonium, thioflavin T, propidium, huprine, and ethopropazine. We compare these structures to equivalent AChE complexes when available in the protein data bank and supplement this comparison with kinetic data and observations from isothermal titration calorimetry. This new information now allows us to define the binding mode of various ligand families and will be of importance in designing specific reversible ligands of BChE that behave as inhibitors or reactivators.
ESTHER : Rosenberry_2017_Molecules_22_
PubMedSearch : Rosenberry_2017_Molecules_22_
PubMedID: 29186056
Gene_locus related to this paper: human-BCHE

Title : Hopeahainol A binds reversibly at the acetylcholinesterase (AChE) peripheral site and inhibits enzyme activity with a novel higher order concentration dependence - Rosenberry_2016_Chem.Biol.Interact_259_78
Author(s) : Rosenberry TL , Martin PK , Nix AJ , Wildman SA , Cheung J , Snyder SA , Tan RX
Ref : Chemico-Biological Interactions , 259 :78 , 2016
Abstract : Natural product inhibitors of AChE are of interest both because they offer promise as inexpensive drugs for symptomatic relief in Alzheimer's disease and because they may provide insights into the structural features of the AChE catalytic site. Hopeahainol A is an uncharged polyphenol AChE inhibitor from the stem bark of H. hainanensis with a constrained, partially dearomatized bicyclic core. Molecular modeling indicates that hopeahainol A binds at the entrance of the long but narrow AChE active site gorge because it is too bulky to be accommodated within the gorge without severe distortion of the gorge as depicted in AChE crystal structures. We conducted inhibitor competition experiments in which AChE inhibition was measured with hopeahainol A together with either edrophonium (which binds at the base of the gorge) or thioflavin T (which binds to the peripheral or P-site near the gorge mouth). The results agreed with the molecular modeling and indicated that hopeahainol A at lower concentrations (<200 muM) bound only to the P-site, as hopeahainol A and thioflavin T were unable to form a ternary complex with AChE while hopeahainol A and edrophonium did form a ternary complex with essentially no competition between them. Inhibition increased to a striking extent at higher concentrations of hopeahainol A, with plots analogous to classic Dixon plots showing a dependence on hopeahainol A concentrations to the third- or fourth order. The inhibition at higher hopeahainol A concentrations was completely reversed on dilution and blocked by bound edrophonium. We hypothesize that bound hopeahainol A induces conformational changes in the AChE active site that allow binding of additional hopeahainol A molecules, a phenomenon that would be unprecedented for a reversible inhibitor that apparently forms no covalent bonds with AChE.
ESTHER : Rosenberry_2016_Chem.Biol.Interact_259_78
PubMedSearch : Rosenberry_2016_Chem.Biol.Interact_259_78
PubMedID: 27297626

Title : Development of acetophenone ligands as potential neuroimaging agents for cholinesterases - Jollymore-Hughes_2016_Bioorg.Med.Chem_24_5270
Author(s) : Jollymore-Hughes CT , Pottie IR , Martin E , Rosenberry TL , Darvesh S
Ref : Bioorganic & Medicinal Chemistry , 24 :5270 , 2016
Abstract : Association of cholinesterase with beta-amyloid plaques and tau neurofibrillary tangles in Alzheimer's disease offers an opportunity to detect disease pathology during life. Achieving this requires development of radiolabelled cholinesterase ligands with high enzyme affinity. Various fluorinated acetophenone derivatives bind to acetylcholinesterase with high affinity, including 2,2,2-trifluoro-1-(3-dimethylaminophenyl)ethanone (1) and 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (2). Such compounds also offer potential for incorporation of radioactive fluorine (18F) for Positron Emission Tomography (PET) imaging of cholinesterases in association with Alzheimer's disease pathology in the living brain. Here we describe the synthesis of two meta-substituted chlorodifluoroacetophenones using a Weinreb amide strategy and their rapid conversion to the corresponding trifluoro derivatives through nucleophilic substitution by fluoride ion, in a reaction amenable to incorporating 18F for PET imaging. In vitro kinetic analysis indicates tight binding of the trifluoro derivatives to cholinesterases. Compound 1 has a Ki value of 7nM for acetylcholinesterase and 1300nM for butyrylcholinesterase while for compound 2 these values are 0.4nM and 26nM, respectively. Tight binding of these compounds to cholinesterase encourages their development for PET imaging detection of cholinesterase associated with Alzheimer's disease pathology.
ESTHER : Jollymore-Hughes_2016_Bioorg.Med.Chem_24_5270
PubMedSearch : Jollymore-Hughes_2016_Bioorg.Med.Chem_24_5270
PubMedID: 27637382

Title : Acetylcholinesterase complexes with the natural product inhibitors dihydrotanshinone I and territrem B: binding site assignment from inhibitor competition and validation through crystal structure determination - Cheung_2014_J.Mol.Neurosci_53_506
Author(s) : Cheung J , Beri V , Shiomi K , Rosenberry TL
Ref : Journal of Molecular Neuroscience , 53 :506 , 2014
Abstract : Acetylcholinesterase (AChE) is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimer's disease and organophosphate (OP) chemical warfare agents that cripple the nervous system and cause death through paralysis. We are exploring a strategy to design compounds that bind tightly at or near a peripheral or P-site near the mouth of the AChE active site gorge and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. Here, we review our recent reports on two uncharged, natural product inhibitors of AChE, dihydrotanshinone I and territrem B, that have relatively high affinities for the enzyme. We describe an inhibitor competition assay and comment on the structures of these inhibitors in complex with recombinant human acetylcholinesterase as determined by X-ray crystallography. Our results reveal that dihydrotanshinone I binding is specific to only the P-site, while territrem B binding spans the P-site and extends into the acylation or A-site at the base of the gorge.
ESTHER : Cheung_2014_J.Mol.Neurosci_53_506
PubMedSearch : Cheung_2014_J.Mol.Neurosci_53_506
PubMedID: 24573600

Title : The natural product dihydrotanshinone I provides a prototype for uncharged inhibitors that bind specifically to the acetylcholinesterase peripheral site with nanomolar affinity - Beri_2013_Biochemistry_52_7486
Author(s) : Beri V , Wildman SA , Shiomi K , Al-Rashid ZF , Cheung J , Rosenberry TL
Ref : Biochemistry , 52 :7486 , 2013
Abstract : Cholinergic synaptic transmission often requires extremely rapid hydrolysis of acetylcholine by acetylcholinesterase (AChE). AChE is inactivated by organophosphates (OPs) in chemical warfare nerve agents. The resulting accumulation of acetylcholine disrupts cholinergic synaptic transmission and can lead to death. A potential long-term strategy for preventing AChE inactivation by OPs is based on evidence that OPs must pass through a peripheral site or P-site near the mouth of the AChE active site gorge before reacting with a catalytic serine in an acylation site or A-site at the base of the gorge. An ultimate goal of this strategy is to design compounds that bind tightly at or near the P-site and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site with ligands and potential drugs that selectively restrict access, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. We apply here an inhibitor competition assay that can correctly determine whether an AChE inhibitor binds to the P-site, the A-site, or both sites. We have used this assay to examine three uncharged, natural product inhibitors of AChE, including aflatoxin B1, dihydrotanshinone I, and territrem B. The first two of these inhibitors are predicted by the competition assay to bind selectively to the P-site, while territrem B is predicted to span both the P- and A-sites. These predictions have recently been confirmed by X-ray crystallography. Dihydrotanshinone I, with an observed binding constant (KI) of 750 nM, provides a good lead compound for the development of high-affinity, uncharged inhibitors with specificity for the P-site.
ESTHER : Beri_2013_Biochemistry_52_7486
PubMedSearch : Beri_2013_Biochemistry_52_7486
PubMedID: 24040835

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

Title : Structures of Human Acetylcholinesterase Bound to Dihydrotanshinone I and Territrem B Show Peripheral Site Flexibility - Cheung_2013_ACS.Med.Chem.Lett_4_1091
Author(s) : Cheung J , Gary EN , Shiomi K , Rosenberry TL
Ref : ACS Med Chem Lett , 4 :1091 , 2013
Abstract : Acetylcholinesterase is a critical enzyme that regulates neurotransmission by degrading the neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for both therapeutic drugs that treat Alzheimers disease and chemical warfare agents that cripple the nervous system and cause death through paralysis. The enzyme has both catalytic and peripheral sites to which inhibitors may bind. Structures of recombinant human acetylcholinesterase in complex with the natural product inhibitors dihydrotanshinone I and territrem B reveal dihydrotanshinone I binding that is specific to only the peripheral site and territrem B binding that spans both sites and distorts the protein backbone in the peripheral site. These inhibitors may function as important molecular templates for therapeutics used for treatment of disease and protection against nerve agents.
ESTHER : Cheung_2013_ACS.Med.Chem.Lett_4_1091
PubMedSearch : Cheung_2013_ACS.Med.Chem.Lett_4_1091
PubMedID: 24900610
Gene_locus related to this paper: human-ACHE

Title : Hydrolysis of low concentrations of the acetylthiocholine analogs acetyl(homo)thiocholine and acetyl(nor)thiocholine by acetylcholinesterase may be limited by selective gating at the enzyme peripheral site - Beri_2013_Chem.Biol.Interact_203_38
Author(s) : Beri V , Auletta JT , Maharvi GM , Wood JF , Fauq AH , Rosenberry TL
Ref : Chemico-Biological Interactions , 203 :38 , 2013
Abstract : Hydrolysis of acetylcholine by acetylcholinesterase (AChE) is extremely rapid, with a second-order hydrolysis rate constant kE (often denoted kcat/KM) that approaches 10(8)M(-1)s(-1). AChE contains a deep active site gorge with two sites of ligand binding, an acylation site (or A-site) containing the catalytic triad at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site is known to contribute to catalytic efficiency with acetylthiocholine (AcSCh) by transiently trapping the substrate in a low affinity complex on its way to the A-site, where a short-lived acyl enzyme intermediate is produced. Here we ask whether the P-site does more than simply trap the substrate but in fact selectively gates entry to the A-site to provide specificity for AcSCh (and acetylcholine) relative to the close structural analogs acetyl(homo)thiocholine (Ac-hSCh, which adds one additional methylene group to thiocholine) and acetyl(nor)thiocholine (Ac-nSCh, which deletes one methylene group from thiocholine). We synthesized Ac-hSCh and Ac-nSCh and overcame technical difficulties associated with instability of the northiocholine hydrolysis product. We then compared the catalytic parameters of these substrates with AChE to those of AcSCh. Values of kE for Ac-hSCh and Ac-nSCh were about 2% of that for AcSCh. The kE for AcSCh is close to the theoretical diffusion-controlled limit for the substrate association rate constant, but kE values for Ac-hSCh or Ac-nSCh are too low to be limited by diffusion control. However, analyses of kinetic solvent isotope effects and inhibition patterns for P-site inhibitors indicate that these two analogs also do not equilibrate with the A-site prior to the initial acylation step of catalysis. We propose that kE for these substrates is partially rate-limited by a gating step that involves the movement of bound substrate from the P-site to the A-site.
ESTHER : Beri_2013_Chem.Biol.Interact_203_38
PubMedSearch : Beri_2013_Chem.Biol.Interact_203_38
PubMedID: 23047027

Title : Probing the peripheral site of human butyrylcholinesterase - Macdonald_2012_Biochemistry_51_7046
Author(s) : Macdonald IR , Martin E , Rosenberry TL , Darvesh S
Ref : Biochemistry , 51 :7046 , 2012
Abstract : Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) catalyze the hydrolysis of the neurotransmitter acetylcholine and, thereby, function as coregulators of cholinergic neurotransmission. For both enzymes, hydrolysis takes place near the bottom of a 20 deep active site gorge. A number of amino acid residues within the gorge have been identified as important in facilitating efficient catalysis and inhibitor binding. Of particular interest is the catalytic triad, consisting of serine, histidine, and glutamate residues, that mediates hydrolysis. Another site influencing the catalytic process is located above the catalytic triad toward the periphery of the active site gorge. This peripheral site (P-site) contains a number of aromatic amino acid residues as well as an aspartate residue that is able to interact with cationic substrates and guide them down the gorge to the catalytic triad. In human AChE, certain aryl residues in the vicinity of the anionic aspartate residue (D74), such as W286, have been implicated in ligand binding and have therefore been considered part of the P-site of the enzyme. The present study was undertaken to explore the P-site of human BuChE and determine whether, like AChE, aromatic side chains near the peripheral aspartate (D70) of this enzyme contribute to ligand binding. Results obtained, utilizing inhibitor competition studies and BuChE mutant species, indicate the participation of aryl residues (F329 and Y332) in the E-helix component of the BuChE active site gorge, along with the anionic aspartate residue (D70), in binding ligands to the P-site of the enzyme.
ESTHER : Macdonald_2012_Biochemistry_51_7046
PubMedSearch : Macdonald_2012_Biochemistry_51_7046
PubMedID: 22901043

Title : Drug-like leads for steric discrimination between substrate and inhibitors of human acetylcholinesterase - Wildman_2011_Chem.Biol.Drug.Des_78_495
Author(s) : Wildman SA , Zheng X , Sept D , Auletta JT , Rosenberry TL , Marshall GR
Ref : Chemical Biology Drug Des , 78 :495 , 2011
Abstract : Protection of the enzyme acetylcholinesterase (AChE) from the toxic effects of organophosphate insecticides and chemical warfare agents (OPs) may be provided by inhibitors that bind at the peripheral binding site (P-site) near the mouth of the active-site gorge. Compounds that bind to this site may selectively block access to the acylation site (A-site) catalytic serine for OPs, but not acetylcholine itself. To identify such compounds, we employed a virtual screening approach using AutoDock 4.2 and AutoDock Vina, confirmed using compounds experimentally known to bind specifically to either the A-site or P-site. Both programs demonstrated the ability to correctly predict the binding site. Virtual screening of the NCI Diversity Set II was conducted using the apo form of the enzyme, and with acetylcholine bound at the crystallographic locations in the A-site only and in both and A- and P-sites. The docking identified 32 compounds that were obtained for testing, and one was demonstrated to bind specifically to the P-site in an inhibitor competition assay.
ESTHER : Wildman_2011_Chem.Biol.Drug.Des_78_495
PubMedSearch : Wildman_2011_Chem.Biol.Drug.Des_78_495
PubMedID: 21668653

Title : Strategies to resolve the catalytic mechanism of acetylcholinesterase - Rosenberry_2010_J.Mol.Neurosci_40_32
Author(s) : Rosenberry TL
Ref : Journal of Molecular Neuroscience , 40 :32 , 2010
Abstract : Acetylcholinesterase (AChE) hydrolyzes its physiological substrate acetylcholine at one of the highest known catalytic rates. Two sites of ligand interaction have been identified: an acylation site or A-site at the base of the active-site gorge and a peripheral site or P-site at its mouth. Although much is known about AChE structure and the role of specific residues in catalysis, a detailed understanding of the catalytic mechanism and the role of the P-site has lagged far behind. In recent years, we have clarified how the P-site and A-site interact to promote catalysis. Our studies revealed that the P-site mediates substrate trapping and that ligand binding to the P-site can result in steric blockade of the A-site as well as allosteric activation of substrate hydrolysis. Because a general, nonequilibrium treatment of AChE catalysis results in complex enzyme kinetic formulations, three simpler, overlapping strategies are presented here that provide significant insights into the AChE catalytic mechanism. The strategies are (1) to choose substrates, preferably close analogs of acetylcholine, that render some intermediates in the general reaction scheme negligible; (2) obtain some of the thermodynamic parameters in this scheme with experiments that are independent of kinetic measurements.
ESTHER : Rosenberry_2010_J.Mol.Neurosci_40_32
PubMedSearch : Rosenberry_2010_J.Mol.Neurosci_40_32
PubMedID: 19757206

Title : Molecular basis of inhibition of substrate hydrolysis by a ligand bound to the peripheral site of acetylcholinesterase - Auletta_2010_Chem.Biol.Interact_187_135
Author(s) : Auletta JT , Johnson JL , Rosenberry TL
Ref : Chemico-Biological Interactions , 187 :135 , 2010
Abstract : Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to the catalytic efficiency of substrate hydrolysis by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Ligands that bind to the A-site invariably inhibit the hydrolysis of all AChE substrates, but ligands that bind to the P-site inhibit the hydrolysis of some substrates but not others. To clarify the basis of this difference, we focus here on second-order rate constants for substrate hydrolysis (k(E)), a parameter that reflects the binding of ligands only to the free form of the enzyme and not to enzyme-substrate intermediates. We first describe an inhibitor competition assay that distinguishes whether a ligand is inhibiting AChE by binding to the A-site or the P-site. We then show that the P-site-specific ligand thioflavin T inhibits the hydrolysis of the rapidly hydrolyzed substrate acetylthiocholine but fails to show any inhibition of the slowly hydrolyzed substrates ATMA (3-(acetamido)-N,N,N-trimethylanilinium) and carbachol. We derive an expression for k(E) that accounts for these observations by recognizing that the rate-limiting steps for these substrates differ. The rate-limiting step for the slow substrates is the general base-catalyzed acylation reaction k(2), a step that is unaffected by bound thioflavin T. In contrast, the rate-limiting step for acetylthiocholine is either substrate association or substrate migration to the A-site, and these steps are blocked by bound thioflavin T.
ESTHER : Auletta_2010_Chem.Biol.Interact_187_135
PubMedSearch : Auletta_2010_Chem.Biol.Interact_187_135
PubMedID: 20493829

Title : Acetylcholinesterase: from 3D structure to function - Dvir_2010_Chem.Biol.Interact_187_10
Author(s) : Dvir H , Silman I , Harel M , Rosenberry TL , Sussman JL
Ref : Chemico-Biological Interactions , 187 :10 , 2010
Abstract : By rapid hydrolysis of the neurotransmitter, acetylcholine, acetylcholinesterase terminates neurotransmission at cholinergic synapses. Acetylcholinesterase is a very fast enzyme, functioning at a rate approaching that of a diffusion-controlled reaction. The powerful toxicity of organophosphate poisons is attributed primarily to their potent inhibition of acetylcholinesterase. Acetylcholinesterase inhibitors are utilized in the treatment of various neurological disorders, and are the principal drugs approved thus far by the FDA for management of Alzheimer's disease. Many organophosphates and carbamates serve as potent insecticides, by selectively inhibiting insect acetylcholinesterase. The determination of the crystal structure of Torpedo californica acetylcholinesterase permitted visualization, for the first time, at atomic resolution, of a binding pocket for acetylcholine. It also allowed identification of the active site of acetylcholinesterase, which, unexpectedly, is located at the bottom of a deep gorge lined largely by aromatic residues. The crystal structure of recombinant human acetylcholinesterase in its apo-state is similar in its overall features to that of the Torpedo enzyme; however, the unique crystal packing reveals a novel peptide sequence which blocks access to the active-site gorge.
ESTHER : Dvir_2010_Chem.Biol.Interact_187_10
PubMedSearch : Dvir_2010_Chem.Biol.Interact_187_10
PubMedID: 20138030
Gene_locus related to this paper: human-ACHE

Title : Assembly and regulation of acetylcholinesterase at the vertebrate neuromuscular junction - Rotundo_2008_Chem.Biol.Interact_175_26
Author(s) : Rotundo RL , Ruiz CA , Marrero E , Kimbell LM , Rossi SG , Rosenberry TL , Darr A , Tsoulfas P
Ref : Chemico-Biological Interactions , 175 :26 , 2008
Abstract : The collagen-tailed form of acetylcholinesterase (ColQ-AChE) is the major if not unique form of the enzyme associated with the neuromuscular junction (NMJ). This enzyme form consists of catalytic and non-catalytic subunits encoded by separate genes, assembled as three enzymatic tetramers attached to the three-stranded collagen-like tail (ColQ). This synaptic form of the enzyme is tightly attached to the basal lamina associated with the glycosaminoglycan perlecan. Fasciculin-2 is a snake toxin that binds tightly to AChE. Localization of junctional AChE on frozen sections of muscle with fluorescent Fasciculin-2 shows that the labeled toxin dissociates with a half-life of about 36 h. The fluorescent toxin can subsequently be taken up by the muscle fibers by endocytosis giving the appearance of enzyme recycling. Newly synthesized AChE molecules undergo a lengthy series of processing events before final transport to the cell surface and association with the synaptic basal lamina. Following co-translational glycosylation the catalytic subunit polypeptide chain interacts with several molecular chaperones, glycosidases and glycosyltransferases to produce a catalytically active enzyme that can subsequently bind to one of two non-catalytic subunits. These molecular chaperones can be rate limiting steps in the assembly process. Treatment of muscle cells with a synthetic peptide containing the PRAD attachment sequence and a KDEL retention signal results in a large increase in assembled and exportable AChE, providing an additional level of post-translational control. Finally, we have found that Pumilio2, a member of the PUF family of RNA-binding proteins, is highly concentrated at the vertebrate neuromuscular junction where it plays an important role in regulating AChE translation through binding to a highly conserved NANOS response element in the 3'-UTR. Together, these studies define several new levels of AChE regulation in electrically excitable cells.
ESTHER : Rotundo_2008_Chem.Biol.Interact_175_26
PubMedSearch : Rotundo_2008_Chem.Biol.Interact_175_26
PubMedID: 18599029

Title : Crystal structure of thioflavin T bound to the peripheral site of Torpedo californica acetylcholinesterase reveals how thioflavin T acts as a sensitive fluorescent reporter of ligand binding to the acylation site - Harel_2008_J.Am.Chem.Soc_130_7856
Author(s) : Harel M , Sonoda LK , Silman I , Sussman JL , Rosenberry TL
Ref : Journal of the American Chemical Society , 130 :7856 , 2008
Abstract : Acetylcholinesterase plays a key role in cholinergic synaptic transmission by hydrolyzing the neurotransmitter acetylcholine with one of the highest known catalytic rate constants. Hydrolysis occurs in a narrow and deep gorge that contains two sites of ligand binding: A peripheral site, or P-site, near the gorge entrance that contributes to catalytic efficiency both by transiently trapping substrate molecules as they enter the gorge and by allosterically accelerating the transfer of the substrate acyl group to a serine hydroxyl in an acylation site or A-site at the base of the gorge. Thioflavin T is a useful reporter of ligand interactions with the A-site. It binds specifically to the P-site with fluorescence that is enhanced approximately 1000-fold over that of unbound thioflavin T, and the enhanced fluorescence is quenched 1.5- to 4-fold when another ligand binds to the A-site in a ternary complex. To clarify the structural basis of this advantageous signal change, we here report the X-ray structure of the complex of thioflavin T with Torpedo californica acetylcholinesterase. The two aromatic rings in thioflavin T are coplanar and are packed snugly parallel to the aromatic side chains of Trp279, Tyr334, and Phe330. Overlays of this structure with the crystal structures of Torpedo californica acetylcholinesterase complexes with either edrophonium or m-( N, N, N-trimethylammonio)-2,2,2-trifluoroacetophenone, two small aromatic ligands that bind specifically to the A-site, indicate that the phenyl side chain of Phe330 must rotate to sterically accommodate both thioflavin T and the A-site ligand in the ternary complex. This rotation may allow some relaxation of the strict coplanarity of the aromatic rings in the bound thioflavin T and result in partial quenching of its fluorescence.
ESTHER : Harel_2008_J.Am.Chem.Soc_130_7856
PubMedSearch : Harel_2008_J.Am.Chem.Soc_130_7856
PubMedID: 18512913
Gene_locus related to this paper: torca-ACHE

Title : Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter - Rosenberry_2008_Biochemistry_47_13056
Author(s) : Rosenberry TL , Sonoda LK , Dekat SE , Cusack B , Johnson JL
Ref : Biochemistry , 47 :13056 , 2008
Abstract : Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acylated enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex with free AChE (E) and proceed to an acylated enzyme intermediate (EC), which is then hydrolyzed. However, the hydrolysis of EC is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here, we focus on the reaction of carbachol (carbamoylcholine) with AChE. The kinetics and thermodynamics of this reaction are of special interest because carbachol is an isosteric analogue of the physiological substrate acetylcholine. We show that the reaction can be monitored with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. Analysis of the fluorescence reaction profiles was challenging because four thermodynamic parameters and two fluorescence coefficients were fitted from the combined data both for E and for EC. Respective equilibrium dissociation constants of 6 and 26 mM were obtained for carbachol binding to the A- and P-sites in E and of 2 and 32 mM for carbachol binding to the A- and P-sites in EC. These constants for the binding of carbachol to the P-site are about an order of magnitude larger (i.e., indicating lower affinity) than previous estimates for the binding of acetylthiocholine to the P-site.
ESTHER : Rosenberry_2008_Biochemistry_47_13056
PubMedSearch : Rosenberry_2008_Biochemistry_47_13056
PubMedID: 19006330

Title : Monitoring the reaction of carbachol with acetylcholinesterase by thioflavin T fluorescence and acetylthiocholine hydrolysis - Rosenberry_2008_Chem.Biol.Interact_175_235
Author(s) : Rosenberry TL , Sonoda LK , Dekat SE , Cusack B , Johnson JL
Ref : Chemico-Biological Interactions , 175 :235 , 2008
Abstract : Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex and proceed to an acylated enzyme intermediate which is then hydrolyzed. However, the hydrolysis of the carbamoylated enzyme is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here we show that the reaction of carbachol (carbamoylcholine) with AChE can be monitored both with acetylthiocholine as a reporter substrate and with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. These fluorescence changes allow not only the monitoring of the course of the carbamoylation reaction but also the determination of carbachol affinities for the A- and P-sites.
ESTHER : Rosenberry_2008_Chem.Biol.Interact_175_235
PubMedSearch : Rosenberry_2008_Chem.Biol.Interact_175_235
PubMedID: 18602908

Title : Interactions between the peripheral site and the acylation site in acetylcholinesterase - Rosenberry_2005_Chem.Biol.Interact_157-158_181
Author(s) : Rosenberry TL , Johnson JL , Cusack B , Thomas JL , Emani S , Venkatasubban KS
Ref : Chemico-Biological Interactions , 157-158 :181 , 2005
Abstract : Acetylcholinesterase (AChE) hydrolyzes its physiological substrate acetylcholine at one of the highest known catalytic rates. Two sites of ligand interaction have been identified: an acylation site or A-site at the base of the active site gorge, and a peripheral site or P-site at its mouth. Despite a wealth of information about the AChE structure and the role of specific residues in catalysis, an understanding of the catalytic mechanism and the role of the P-site has lagged far behind. In recent years we have clarified how the P- and A-sites interact to promote catalysis. Our studies have revealed that the P-site mediates substrate trapping and that ligand binding to the P-site can result in steric blockade of the A-site as well as allosteric activation. We have demonstrated this activation only for the acylation step of the catalytic reaction, but others have proposed that it involves the deacylation step. To investigate this point, we have measured the reaction of carbamoyl esters (carbamates) with AChE. With these slowly hydrolyzed substrates, the carbamoylation (acylation) and decarbamoylation (deacylation) steps can be resolved and analyzed separately. Carbamoylcholine is one of the closest structural analogs of acetylcholine, and we monitored these steps in continuous mixed assays with acetylthiocholine as a reporter substrate. At high concentrations of carbamoylcholine, decarbamoylation was inhibited but no activation of carbamoylation was observed. However, high concentrations of acetylthiocholine had no effect on the decarbamoylation rate constants. We concluded that the binding of acetylthiocholine to the P-site does not activate deacylation reactions.
ESTHER : Rosenberry_2005_Chem.Biol.Interact_157-158_181
PubMedSearch : Rosenberry_2005_Chem.Biol.Interact_157-158_181
PubMedID: 16256966

Title : A novel strategy for protection against organophosphate toxicity: Evolution of cyclic inhibitors with high affinity for the acetylcholinesterase peripheral site -
Author(s) : Cusack B , Romanovskis P , Johnson JL , Etienne G , Rosenberry TL
Ref : Chemico-Biological Interactions , 157-158 :370 , 2005
PubMedID: 16429491

Title : Poster (76) Modulation of acetylcholinesterase kinetics employing ligands tethered to the mutant H287C: a model for organophosphate inhibitor desin -
Author(s) : Cusack B , Johnson JL , Hughes TF , McCullough EH , Romanovskis PV , Spatola AF , Rosenberry TL
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :360 , 2004
PubMedID:

Title : Tethering of ligands near the active site of acetylcholinesterase mutant H287C: Progress on a new strategy for protection against organophosphate inactivation -
Author(s) : Cusack B , Johnson JL , Hughes TF , McCullough EH , Fauq A , Romanovskis PV , Spatola AF , Rosenberry TL
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :259 , 2004
PubMedID:

Title : Poster (87) Ligand interactions within the active site of acetylcholinesterase -
Author(s) : Johnson JL , Cusack B , Davies MP , Rosenberry TL
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :366 , 2004
PubMedID:

Title : Poster (16) Ligand interactions within the active site of acetylcholinesterase -
Author(s) : Johnson JL , Cusack B , Davies MP , Rosenberry TL
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :328 , 2004
PubMedID:

Title : Poster (17) Short, strong hydrogen bonds at the active site of cholinesterases: H-NMR studies. -
Author(s) : Kovach IM , Viragh C , Reddy PM , Massiah MA , Mildvan AS , Johnson JL , Rosenberry TL
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :329 , 2004
PubMedID:

Title : Substrate activation with a cationic acetanilide substrate in human acetylcholinesterase -
Author(s) : Johnson JL , Cusack B , Davies MP , Fauq A , Rosenberry TL
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :213 , 2004
PubMedID:

Title : Mechanisms of altered vagal control in heart failure: influence of muscarinic receptors and acetylcholinesterase activity - Dunlap_2003_Am.J.Physiol.Heart.Circ.Physiol_285_H1632
Author(s) : Dunlap ME , Bibevski S , Rosenberry TL , Ernsberger P
Ref : American Journal of Physiology Heart Circ Physiol , 285 :H1632 , 2003
Abstract : Parasympathetic control of the heart is attenuated in heart failure (HF). We investigated possible mechanisms and sites of altered vagal control in dogs with HF induced by rapid pacing. Muscarinic blockade reduced the R-R interval by 308 ms in controls but only by 32 ms in HF, indicating low levels of resting vagal tone. Vagomimetic doses of atropine sulfate prolonged the R-R interval by 109 ms in controls and increased standard deviation of the R-R interval by 66 ms but only by 46 and 16 ms, respectively, in HF. Bradycardia elicited by electrical stimulation of the vagus nerve was also attenuated in the HF group. Conversely, muscarinic receptor activation by bethanechol, and indirectly by neostigmine, elicited exaggerated R-R interval responses in HF. To investigate possible mechanisms, we measured muscarinic receptor density (Bmax) and acetylcholinesterase activity in different areas of the heart. In sinoatrial nodes, Bmax was increased (230 +/- 75% of control) and acetylcholinesterase decreased (80 +/- 6% of control) in HF. We conclude that muscarinic receptors are upregulated and acetylcholinesterase is reduced in the sinus node in HF. Therefore, reduced vagal control in HF is most likely due to changes of presynaptic function (ganglionic), because postsynaptic mechanisms augment vagal control in HF.
ESTHER : Dunlap_2003_Am.J.Physiol.Heart.Circ.Physiol_285_H1632
PubMedSearch : Dunlap_2003_Am.J.Physiol.Heart.Circ.Physiol_285_H1632
PubMedID: 12829433

Title : Inhibitors tethered near the acetylcholinesterase active site serve as molecular rulers of the peripheral and acylation sites - Johnson_2003_J.Biol.Chem_278_38948
Author(s) : Johnson JL , Cusack B , Hughes TF , McCullough EH , Fauq A , Romanovskis P , Spatola AF , Rosenberry TL
Ref : Journal of Biological Chemistry , 278 :38948 , 2003
Abstract : The acetylcholinesterase (AChE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C AChE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants KI2 for propidium and tacrine, inhibitors specific for the P- and A-sites, respectively. Values of KI2 for propidium increased 30- to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of KI2 for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tacrine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations.
ESTHER : Johnson_2003_J.Biol.Chem_278_38948
PubMedSearch : Johnson_2003_J.Biol.Chem_278_38948
PubMedID: 12851386

Title : Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate - Johnson_2003_Biochemistry_42_5438
Author(s) : Johnson JL , Cusack B , Davies MP , Fauq A , Rosenberry TL
Ref : Biochemistry , 42 :5438 , 2003
Abstract : Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate constants in the initial intermediate formed during acylation (EAP, where EA is the acyl enzyme and P is the alcohol leaving group cleaved from the ester substrate) may be constrained such that the leaving group P must dissociate before hydrolytic deacylation can occur.
ESTHER : Johnson_2003_Biochemistry_42_5438
PubMedSearch : Johnson_2003_Biochemistry_42_5438
PubMedID: 12731886

Title : 3D structure of Torpedo californica acetylcholinesterase complexed with huprine X at 2.1 A resolution: kinetic and molecular dynamic correlates - Dvir_2002_Biochemistry_41_2970
Author(s) : Dvir H , Wong DM , Harel M , Barril X , Orozco M , Luque FJ , Munoz-Torrero D , Camps P , Rosenberry TL , Silman I , Sussman JL
Ref : Biochemistry , 41 :2970 , 2002
Abstract : Huprine X is a novel acetylcholinesterase (AChE) inhibitor, with one of the highest affinities reported for a reversible inhibitor. It is a synthetic hybrid that contains the 4-aminoquinoline substructure of one anti-Alzheimer drug, tacrine, and a carbobicyclic moiety resembling that of another AChE inhibitor, (-)-huperzine A. Cocrystallization of huprine X with Torpedo californica AChE yielded crystals whose 3D structure was determined to 2.1 A resolution. The inhibitor binds to the anionic site and also hinders access to the esteratic site. Its aromatic portion occupies the same binding site as tacrine, stacking between the aromatic rings of Trp84 and Phe330, whereas the carbobicyclic unit occupies the same binding pocket as (-)-huperzine A. Its chlorine substituent was found to lie in a hydrophobic pocket interacting with rings of the aromatic residues Trp432 and Phe330 and with the methyl groups of Met436 and Ile439. Steady-state inhibition data show that huprine X binds to human AChE and Torpedo AChE 28- and 54-fold, respectively, more tightly than tacrine. This difference stems from the fact that the aminoquinoline moiety of huprine X makes interactions similar to those made by tacrine, but additional bonds to the enzyme are made by the huperzine-like substructure and the chlorine atom. Furthermore, both tacrine and huprine X bind more tightly to Torpedo than to human AChE, suggesting that their quinoline substructures interact better with Phe330 than with Tyr337, the corresponding residue in the human AChE structure. Both (-)-huperzine A and huprine X display slow binding properties, but only binding of the former causes a peptide flip of Gly117.
ESTHER : Dvir_2002_Biochemistry_41_2970
PubMedSearch : Dvir_2002_Biochemistry_41_2970
PubMedID: 11863435
Gene_locus related to this paper: torca-ACHE

Title : Properties of exogenously added GPI-anchored proteins following their incorporation into cells - Premkumar_2001_J.Cell.Biochem_82_234
Author(s) : Premkumar DR , Fukuoka Y , Sevlever D , Brunschwig E , Rosenberry TL , Tykocinski ML , Medof ME
Ref : Journal of Cellular Biochemistry , 82 :234 , 2001
Abstract : Isolated glycosylphosphatidylinositol (GPI)-anchored proteins, when added to cells in vitro, incorporate into their surface membranes and, once incorporated, exert their native functions. Virtually any protein of interest, if expressed as a GPI-reanchored derivative, can be modified to acquire this capacity. Such transfer of proteins directly to cells, termed "protein engineering" or "painting" constitutes an alternative to conventional gene transfer for manipulating cell surface composition that has many potential applications. Previous studies with incorporated GPI-anchored proteins have focused almost entirely on their extracellular functions. In this study, biotinylated human erythrocyte (E(hu)) decay accelerating factor, E(hu) acetylcholinesterase, and GPI-reanchored murine B7-1 and B7-2 were used as GPI-anchored reporters to characterize their plasma membrane organization and cell signalling properties following addition to Hela or Chinese hamster ovary cells. For each reporter, three types of cell-association were documented; (1) nonphysiological attachment and/or incomplete insertion, (2) uncomplexed membrane integration, and (3) organization into TX-100-resistant microdomains. Transit from the first two compartments into the third, i.e., microdomains, progressed slowly, continuing even after 24 to 36 h and was associated with the acquisition of cell signalling capacity. All four reporters, incorporated in two different detergents, behaved similarly. When organized in microdomains, caveolin and other GPI proteins co-isolated with the incorporated reporter. These results have implications for protein engineering of cells in general, and in particular, for cells such as modified tumor cell immunogens administered to patients for therapeutic purposes.
ESTHER : Premkumar_2001_J.Cell.Biochem_82_234
PubMedSearch : Premkumar_2001_J.Cell.Biochem_82_234
PubMedID: 11527149

Title : Short, Strong Hydrogen Bonds at the Active Site of Human Acetylcholinesterase: Proton NMR Studies - Massiah_2001_Biochemistry_40_5682
Author(s) : Massiah MA , Viragh C , Reddy PM , Kovach IM , Johnson J , Rosenberry TL , Mildvan AS
Ref : Biochemistry , 40 :5682 , 2001
Abstract : Cholinesterases use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. We have previously shown by proton NMR that horse serum butyryl cholinesterase, like serine proteases, forms a short, strong hydrogen bond (SSHB) between the Glu-His pair upon binding mechanism-based inhibitors, which form tetrahedral adducts, analogous to the tetrahedral intermediates in catalysis [Viragh, C., et al. (2000) Biochemistry 39, 16200-16205]. We now extend these studies to human acetylcholinesterase, a 136 kDa homodimer. The free enzyme at pH 7.5 shows a proton resonance at 14.4 ppm assigned to an imidazole NH of the active-site histidine, but no deshielded proton resonances between 15 and 21 ppm. Addition of a 3-fold excess of the mechanism-based inhibitor m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) induced the complete loss of the 14.4 ppm signal and the appearance of a broad, deshielded resonance of equal intensity with a chemical shift delta of 17.8 ppm and a D/H fractionation factor phi of 0.76 +/- 0.10, consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen bond lengths in small crystalline compounds, the length of this SSHB is 2.62 +/- 0.02 A, in agreement with the length of 2.63 +/- 0.03 A, independently obtained from phi. Upon addition of a 3-fold excess of the mechanism-based inhibitor 4-nitrophenyl diethyl phosphate (paraoxon) to the free enzyme at pH 7.5, and subsequent deethylation, two deshielded resonances of unequal intensity appeared at 16.6 and 15.5 ppm, consistent with SSHBs with lengths of 2.63 +/- 0.02 and 2.65 +/- 0.02 A, respectively, suggesting conformational heterogeneity of the active-site histidine as a hydrogen bond donor to either Glu-327 of the catalytic triad or to Glu-199, also in the active site. Conformational heterogeneity was confirmed with the methylphosphonate ester anion adduct of the active-site serine, which showed two deshielded resonances of equal intensity at 16.5 and 15.8 ppm with phi values of 0.47 +/- 0.10 and 0.49 +/- 0.10 corresponding to average hydrogen bond lengths of 2.59 +/- 0.04 and 2.61 +/- 0.04 A, respectively. Similarly, lowering the pH of the free enzyme to 5.1 to protonate the active-site histidine (pK(a) = 6.0 +/- 0.4) resulted in the appearance of two deshielded resonances, at 17.7 and 16.4 ppm, consistent with SSHBs with lengths of 2.62 +/- 0.02 and 2.63 +/- 0.02 A, respectively. The NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase from Torpedo californica complexed with TMTFA (2.66 +/- 0.28 A) and sarin (2.53 +/- 0.26 A) and at low pH (2.52 +/- 0.25 A). However, the order of magnitude greater precision of the NMR-derived distances establishes the presence of SSHBs at the active site of acetylcholinesterase, and detect conformational heterogeneity of the active-site histidine. We suggest that the high catalytic power of cholinesterases results in part from the formation of a SSHB between Glu and His of the catalytic triad.
ESTHER : Massiah_2001_Biochemistry_40_5682
PubMedSearch : Massiah_2001_Biochemistry_40_5682
PubMedID: 11341833

Title : Thioflavin T is a fluorescent probe of the acetylcholinesterase peripheral site that reveals conformational interactions between the peripheral and the acylation sites - De Ferrari_2001_J.Biol.Chem_276_23282
Author(s) : De Ferrari GV , Mallender WD , Inestrosa NC , Rosenberry TL
Ref : Journal of Biological Chemistry , 276 :23282 , 2001
Abstract : Three-dimensional structures of acetylcholinesterase (AChE) reveal a narrow and deep active site gorge with two sites of ligand binding, an acylation site at the base of the gorge, and a peripheral site near the gorge entrance. Recent studies have shown that the peripheral site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, but the question of whether the peripheral site makes other contributions to the catalytic process remains open. A possible role for ligand binding to the peripheral site that has long been considered is the initiation of a conformational change that is transmitted allosterically to the acylation site to alter catalysis. However, evidence for conformational interactions between these sites has been difficult to obtain. Here we report that thioflavin T, a fluorophore widely used to detect amyloid structure in proteins, binds selectively to the AChE peripheral site with an equilibrium dissociation constant of 1.0 microm. The fluorescence of the bound thioflavin T is increased more than 1000-fold over that of unbound thioflavin T, the greatest enhancement of fluorescence for the binding of a fluorophore to AChE yet observed. Furthermore, when the acylation site ligands edrophonium or m-(N, N,N-trimethylammonio)trifluoroacetophenone form ternary complexes with AChE and thioflavin T, the fluorescence is quenched by factors of 2.7-4.2. The observation of this partial quenching of thioflavin T fluorescence is a major advance in the study of AChE for two reasons. First, it allows thioflavin T to be used as a reporter for ligand reactions at the acylation site. Second, it indicates that ligand binding to the acylation site initiates a change in the local AChE conformation at the peripheral site that quenches the fluorescence of bound thioflavin T. The data provide strong evidence in support of a conformational interaction between the two AChE sites.
ESTHER : De Ferrari_2001_J.Biol.Chem_276_23282
PubMedSearch : De Ferrari_2001_J.Biol.Chem_276_23282
PubMedID: 11313335

Title : Acetylthiocholine binds to asp74 at the peripheral site of human acetylcholinesterase as the first step in the catalytic pathway - Mallender_2000_Biochemistry_39_7753
Author(s) : Mallender WD , Szegletes T , Rosenberry TL
Ref : Biochemistry , 39 :7753 , 2000
Abstract : Studies of ligand binding to acetylcholinesterase (AChE) have demonstrated two sites of interaction. An acyl-enzyme intermediate is formed at the acylation site, and catalytic activity can be inhibited by ligand binding to a peripheral site. The three-dimensional structures of AChE-ligand complexes reveal a narrow and deep active site gorge and indicate that ligands specific for the acylation site at the base of the gorge must first traverse the peripheral site near the gorge entrance. In recent studies attempting to clarify the role of the peripheral site in the catalytic pathway for AChE, we showed that ligands which bind specifically to the peripheral site can slow the rates at which other ligands enter and exit the acylation site, a feature we called steric blockade [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. We also demonstrated that cationic substrates can form a low-affinity complex at the peripheral site that accelerates catalytic hydrolysis at low substrate concentrations but results in substrate inhibition at high concentrations because of steric blockade of product release [Szegletes, T., Mallender, W. D., Thomas, P. J., and Rosenberry, T. L. (1999) Biochemistry 38, 122-133]. In this report, we demonstrate that a key residue in the human AChE peripheral site with which the substrate acetylthiocholine interacts is D74. We extend our kinetic model to evaluate the substrate affinity for the peripheral site, indicated by the equilibrium dissociation constant K(S), from the dependence of the substrate hydrolysis rate on substrate concentration. For human AChE, a K(S) of 1.9+/-0.7 mM obtained by fitting this substrate inhibition curve agreed with a K(S) of 1.3+/-1.0 mM measured directly from acetylthiocholine inhibition of the binding of the neurotoxin fasciculin to the peripheral site. For Torpedo AChE, a K(S) of 0.5+/- 0.2 mM obtained from substrate inhibition agreed with a K(S) of 0.4+/- 0.2 mM measured with fasciculin. Introduction of the D72G mutation (corresponding to D74G in human AChE) increased the K(S) to 4-10 mM in the Torpedo enzyme and to about 33 mM in the human enzyme. While the turnover number k(cat) was unchanged in the human D74G mutant, the roughly 20-fold decrease in acetylthiocholine affinity for the peripheral site in D74G resulted in a corresponding decrease in k(cat)/K(app), the second-order hydrolysis rate constant, in the mutant. In addition, we show that D74 is important in conveying to the acylation site an inhibitory conformational effect induced by the binding of fasciculin to the peripheral site. This inhibitory effect, measured by the relative decrease in the first-order phosphorylation rate constant k(OP) for the neutral organophosphate 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC) that resulted from fasciculin binding, decreased from 0.002 in wild-type human AChE to 0.24 in the D74G mutant.
ESTHER : Mallender_2000_Biochemistry_39_7753
PubMedSearch : Mallender_2000_Biochemistry_39_7753
PubMedID: 10869180

Title : Huprine X is a Novel High-Affinity Inhibitor of Acetylcholinesterase That Is of Interest for Treatment of Alzheimer's Disease - Camps_2000_Mol.Pharmacol_57_409
Author(s) : Camps P , Cusack B , Mallender WD , Achab RE , Morral J , Munoz-Torrero D , Rosenberry TL
Ref : Molecular Pharmacology , 57 :409 , 2000
Abstract : Inhibitors of the enzyme acetylcholinesterase (AChE) slow and sometimes reverse the cognitive decline experienced by individuals with Alzheimer's disease. Huperzine A, a natural product used in traditional Chinese herbal medicine, and tacrine (Cognex) are among the potent AChE inhibitors used in this treatment, but the search for more selective inhibitors continues. We report herein the synthesis and characterization of (-)-12-amino-3-chloro-9-ethyl-6,7, 10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride (huprine X), a hybrid that combines the carbobicyclic substructure of huperzine A with the 4-aminoquinoline substructure of tacrine. Huprine X inhibited human AChE with an inhibition constant K(I) of 26 pM, indicating that it binds to this enzyme with one of the highest affinities yet reported. Under equivalent assay conditions, this affinity was 180 times that of huperzine A, 1200 times that of tacrine, and 40 times that of E2020 (donepezil, Aricept), the most selective AChE inhibitor currently approved for therapeutic use. The association and dissociation rate constants for huprine X with AChE were determined, and the location of its binding site on the enzyme was probed in competition studies with the peripheral site inhibitor propidium and the acylation site inhibitor edrophonium. Huprine X showed no detectable affinity for the edrophonium-AChE complex. In contrast, huprine X did form a ternary complex with propidium and AChE, although its affinity for the free enzyme was found to be 17 times its affinity for the propidium-AChE complex. These data indicated that huprine X binds to the enzyme acylation site in the active site gorge but interferes slightly with the binding of peripheral site ligands.
ESTHER : Camps_2000_Mol.Pharmacol_57_409
PubMedSearch : Camps_2000_Mol.Pharmacol_57_409
PubMedID: 10648652

Title : Two distinct proteins are associated with tetrameric acetylcholinesterase on the cell surface - Perrier_2000_J.Biol.Chem_275_34260
Author(s) : Perrier AL , Cousin X , Boschetti N , Haas R , Chatel JM , Bon S , Roberts WL , Pickett SR , Massoulie J , Rosenberry TL , Krejci E
Ref : Journal of Biological Chemistry , 275 :34260 , 2000
Abstract : In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.
ESTHER : Perrier_2000_J.Biol.Chem_275_34260
PubMedSearch : Perrier_2000_J.Biol.Chem_275_34260
PubMedID: 10954708

Title : Three-dimensional structures of Drosophila melanogaster acetylcholinesterase and of its complexes with two potent inhibitors - Harel_2000_Protein.Sci_9_1063
Author(s) : Harel M , Kryger G , Rosenberry TL , Mallender WD , Lewis T , Fletcher RJ , Guss JM , Silman I , Sussman JL
Ref : Protein Science , 9 :1063 , 2000
Abstract : We have crystallized Drosophila melanogaster acetylcholinesterase and solved the structure of the native enzyme and of its complexes with two potent reversible inhibitors, 1,2,3,4-tetrahydro-N-(phenylmethyl)-9-acridinamine and 1,2,3,4-tetrahydro-N-(3-iodophenyl-methyl)-9-acridinamine--all three at 2.7 A resolution. The refined structure of D. melanogaster acetylcholinesterase is similar to that of vertebrate acetylcholinesterases, for example, human, mouse, and fish, in its overall fold, charge distribution, and deep active-site gorge, but some of the surface loops deviate by up to 8 A from their position in the vertebrate structures, and the C-terminal helix is shifted substantially. The active-site gorge of the insect enzyme is significantly narrower than that of Torpedo californica AChE, and its trajectory is shifted several angstroms. The volume of the lower part of the gorge of the insect enzyme is approximately 50% of that of the vertebrate enzyme. Upon binding of either of the two inhibitors, nine aromatic side chains within the active-site gorge change their conformation so as to interact with the inhibitors. Some differences in activity and specificity between the insect and vertebrate enzymes can be explained by comparison of their three-dimensional structures.
ESTHER : Harel_2000_Protein.Sci_9_1063
PubMedSearch : Harel_2000_Protein.Sci_9_1063
PubMedID: 10892800
Gene_locus related to this paper: drome-ACHE

Title : Organophosphorylation of acetylcholinesterase in the presence of peripheral site ligands. Distinct effects of propidium and fasciculin - Mallender_1999_J.Biol.Chem_274_8491
Author(s) : Mallender WD , Szegletes T , Rosenberry TL
Ref : Journal of Biological Chemistry , 274 :8491 , 1999
Abstract : Structural analysis of acetylcholinesterase (AChE) has revealed two sites of ligand interaction in the active site gorge: an acylation site at the base of the gorge and a peripheral site at its mouth. A goal of our studies is to understand how ligand binding to the peripheral site alters the reactivity of substrates and organophosphates at the acylation site. Kinetic rate constants were determined for the phosphorylation of AChE by two fluorogenic organophosphates, 7-[(diethoxyphosphoryl)oxy]-1-methylquinolinium iodide (DEPQ) and 7-[(methylethoxyphosphonyl)oxy]-4-methylcoumarin (EMPC), by monitoring release of the fluorescent leaving group. Rate constants obtained with human erythrocyte AChE were in good agreement with those obtained for recombinant human AChE produced from a high level Drosophila S2 cell expression system. First-order rate constants kOP were 1,600 +/- 300 min-1 for DEPQ and 150 +/- 11 min-1 for EMPC, and second-order rate constants kOP/KOP were 193 +/- 13 microM-1 min-1 for DEPQ and 0.7-1.0 +/- 0.1 microM-1 min-1 for EMPC. Binding of the small ligand propidium to the AChE peripheral site decreased kOP/KOP by factors of 2-20 for these organophosphates. Such modest inhibitory effects are consistent with our recently proposed steric blockade model (Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216). Moreover, the binding of propidium resulted in a clear increase in kOP for EMPC, suggesting that molecular or electronic strain caused by the proximity of propidium to EMPC in the ternary complex may promote phosphorylation. In contrast, the binding of the polypeptide neurotoxin fasciculin to the peripheral site of AChE dramatically decreased phosphorylation rate constants. Values of kOP/KOP were decreased by factors of 10(3) to 10(5), and kOP was decreased by factors of 300-4,000. Such pronounced inhibition suggested a conformational change in the acylation site induced by fasciculin binding. As a note of caution to other investigators, measurements of phosphorylation of the fasciculin-AChE complex by AChE inactivation gave misleading rate constants because a small fraction of the AChE was resistant to inhibition by fasciculin.
ESTHER : Mallender_1999_J.Biol.Chem_274_8491
PubMedSearch : Mallender_1999_J.Biol.Chem_274_8491
PubMedID: 10085081

Title : Substrate binding to the peripheral site of acetylcholinesterase initiates enzymatic catalysis. Substrate inhibition arises as a secondary effect - Szegletes_1999_Biochemistry_38_122
Author(s) : Szegletes T , Mallender WD , Thomas PJ , Rosenberry TL
Ref : Biochemistry , 38 :122 , 1999
Abstract : Two sites of ligand interaction in acetylcholinesterase (AChE) were first demonstrated in ligand binding studies and later confirmed by crystallography, site-specific mutagenesis, and molecular modeling: an acylation site at the base of the active site gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated how small peripheral site ligands such as propidium may inhibit substrate hydrolysis [Szegletes, T., Mallender, W. D., and Rosenberry, T. L. (1998) Biochemistry 37, 4206-4216]. In this model, the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Here, we first provide evidence that not only rate constants for substrates but also dissociation rate constants for their hydrolysis products are decreased by bound peripheral site ligand. Previous reaction schemes for substrate hydrolysis by AChE were extended to include product dissociation steps, and acetylthiocholine hydrolysis rates in the presence of propidium under nonequilibrium conditions were simulated with assigned rate constants in the program SCoP. We next showed that cationic substrates such as acetylthiocholine and 7-acetoxy-N-methylquinolinium (M7A) bind to the peripheral site as well as to the acylation site. The neurotoxin fasciculin was used to report specifically on interactions at the peripheral site. Analysis of inhibition of fasciculin association rates by these substrates revealed KS values of about 1 mM for the peripheral site binding of acetylthiocholine and 0.2 mM for the binding of M7A. The AChE reaction scheme was further extended to include substrate binding to the peripheral site as the initial step in the catalytic pathway. Simulations of the steric blockade model with this scheme were in reasonable agreement with observed substrate inhibition for acetylthiocholine and M7A and with mutual competitive inhibition in mixtures of acetylthiocholine and M7A. Substrate inhibition was explained by blockade of product dissociation when substrate is bound to the peripheral site. However, our analyses indicate that the primary physiologic role of the AChE peripheral site is to accelerate the hydrolysis of acetylcholine at low substrate concentrations.
ESTHER : Szegletes_1999_Biochemistry_38_122
PubMedSearch : Szegletes_1999_Biochemistry_38_122
PubMedID: 9890890

Title : A steric blockade model for inhibition of acetylcholinesterase by peripheral site ligands and substrate - Rosenberry_1999_Chem.Biol.Interact_119-120_85
Author(s) : Rosenberry TL , Mallender WD , Thomas PJ , Szegletes T
Ref : Chemico-Biological Interactions , 119-120 :85 , 1999
Abstract : The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge and a peripheral site at its mouth. We recently introduced a steric blockade model which demonstrated that small peripheral site ligands like propidium can inhibit substrate hydrolysis simply by decreasing the substrate association and dissociation rate constants without altering the equilibrium constant for substrate binding to the acylation site. We now employ our nonequilibrium kinetic analysis to extend this model to include blockade of the dissociation of substrate hydrolysis products by bound peripheral site ligand. We also report here that acetylthiocholine can bind to the AChE peripheral site with an equilibrium dissociation constant K(S) of about 1 mM. This value was determined from the effect of the acetylthiocholine concentration on the rate at which fasciculin associates with the peripheral site. When substrate binding to the peripheral site is incorporated into our steric blockade model, hydrolysis rates at low substrate concentration appear to be accelerated while substrate inhibition of hydrolysis occurs at high substrate concentration. The model predicts that hydrolysis rates for substrates which equilibrate with the acylation site prior to the acylation step should not be inhibited by bound peripheral site ligand. Organophosphates equilibrate with AChE prior to phosphorylating the active site serine residue, and as predicted propidium had little effect on the phosphorylation rate constants for the fluorogenic organophosphate ethylmethyl-phosphonylcoumarin (EMPC). The 2nd-order phosphorylation rate constant kOP/K(OP) was decreased 3-fold by a high concentration of propidium and the 1st-order rate constant kOP increased somewhat. In contrast to propidium, when the neurotoxin fasciculin bound to the AChE peripheral site both a steric blockade and a conformational change in the acylation site appeared to occur. With saturating fasciculin, kOP/K(OP) decreased by a factor of more than 750 and kOP decreased 300-fold. These data suggest that new peripheral site ligands may be designed to have selective effects on AChE phosphorylation.
ESTHER : Rosenberry_1999_Chem.Biol.Interact_119-120_85
PubMedSearch : Rosenberry_1999_Chem.Biol.Interact_119-120_85
PubMedID: 10421442

Title : Nonequilibrium analysis alters the mechanistic interpretation of inhibition of acetylcholinesterase by peripheral site ligands - Szegletes_1998_Biochemistry_37_4206
Author(s) : Szegletes T , Mallender WD , Rosenberry TL
Ref : Biochemistry , 37 :4206 , 1998
Abstract : The active site gorge of acetylcholinesterase (AChE) contains two sites of ligand binding, an acylation site near the base of the gorge with a catalytic triad characteristic of serine hydrolases, and a peripheral site at the mouth of the gorge some 10-20 A from the acylation site. Many ligands that bind exclusively to the peripheral site inhibit substrate hydrolysis at the acylation site, but the mechanistic interpretation of this inhibition has been unclear. Previous interpretations have been based on analyses of inhibition patterns obtained from steady-state kinetic models that assume equilibrium ligand binding. These analyses indicate that inhibitors bound to the peripheral site decrease acylation and deacylation rate constants and/or decrease substrate affinity at the acylation site by factors of up to 100. Conformational interactions have been proposed to account for such large inhibitory effects transmitted over the distance between the two sites, but site-specific mutagenesis has failed to reveal residues that mediate the proposed conformational linkage. Since examination of individual rate constants in the AChE catalytic pathway reveals that assumptions of equilibrium ligand binding cannot be justified, we introduce here an alternative nonequilibrium analysis of the steady-state inhibition patterns. This analysis incorporates a steric blockade hypothesis which assumes that the only effect of a bound peripheral site ligand is to decrease the association and dissociation rate constants for an acylation site ligand without altering the equilibrium constant for ligand binding to the acylation site. Simulations based on this nonequilibrium steric blockade model were in good agreement with experimental data for inhibition by the peripheral site ligands propidium and gallamine at low concentrations of either acetylthiocholine or phenyl acetate if binding of these ligands slows substrate association and dissociation rate constants by factors of 5-70. Direct measurements with the acylation site ligands huperzine A and m-(N,N, N-trimethylammonio)trifluoroacetophenone showed that bound propidium decreased the association rate constants 49- and 380-fold and the dissociation rate constants 10- and 60-fold, respectively, relative to the rate constants for these acylation site ligands with free AChE, in reasonable agreement with the nonequilibrium steric blockade model. We conclude that this model can account for the inhibition of AChE by small peripheral site ligands such as propidium without invoking any conformational interaction between the peripheral and acylation sites.
ESTHER : Szegletes_1998_Biochemistry_37_4206
PubMedSearch : Szegletes_1998_Biochemistry_37_4206
PubMedID: 9521743

Title : Substrate Binding to the Peripheral Site Occurs on the Catalytic Pathway of Acetylcholinesterase and Leads to Substrate Inhibition -
Author(s) : Rosenberry TL , Mallender WD , Thomas PJ , Szegletes T
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. :189 , 1998
PubMedID:

Title : Substrate Binding to the Acetylcholinesterase Peripheral Site Promotes Substrate Hydrolysis but also Gives Rise to Substrate Inhibition -
Author(s) : Szegletes T , Mallender WD , Rosenberry TL
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. :229 , 1998
PubMedID:

Title : Cyclic, Selectively Permeable Acetylcholinesterase Inhibitors -
Author(s) : Mallender WD , Agarwal S , Ma W , Spatola AF , Rosenberry TL
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. :245 , 1998
PubMedID:

Title : Construction and characterization of secreted and chimeric transmembrane forms of Drosophila acetylcholinesterase: a large truncation of the C-terminal signal peptide does not eliminate glycoinositol phospholipid anchoring - Incardona_1996_Mol.Biol.Cell_7_595
Author(s) : Incardona JP , Rosenberry TL
Ref : Molecular Biology of the Cell , 7 :595 , 1996
Abstract : Despite advances in understanding the cell biology of glycoinositol phospholipid (GPI)-anchored proteins in cultured cells, the in vivo functions of GPI anchors have remained elusive. We have focused on Drosophila acetylcholinesterase (AChE) as a model GPI-anchored protein that can be manipulated in vivo with sophisticated genetic techniques. In Drosophila, AChE is found only as a GPI-anchored G2 form encoded by the Ace locus on the third chromosome. To pursue our goal of replacing wild-type GPI-anchored AChE with forms that have alternative anchor structures in transgenic files, we report the construction of two secreted forms of Drosophila AChE (SEC1 and SEC2) and a chimeric form (TM-AChE) anchored by the transmembrane and cytoplasmic domains of herpes simplex virus type 1 glycoprotein C. To confirm that the biochemical properties of these AChEs were unchanged from GPI-AChE except as predicted, we made stably transfected Drosophila Schneider Line 2(S2) cells expressing each of the four forms. TM-AChE, SEC1, and SEC2 had the same catalytic activity and quaternary structure as wild type. TM-AChE was expressed as an amphiphilic membrane-bound protein resistant to an enzyme that cleaves GPI-AChE (phosphatidylinositol-specific phospholipase C), and the same percentage of TM-AChE and GPI-AChE was on the cell surface according to immunofluorescence and pharmacological data. SEC1 and SEC2 were constructed by truncating the C-terminal signal peptide initially present in GPI-AChE: in SEC1 the last 25 residues of this 34-residue peptide were deleted while in SEC2 the last 29 were deleted. Both SEC1 and SEC2 were efficiently secreted and are very stable in culture medium; with one cloned SEC1-expressing line, AChE accumulated to as high as 100 mg/liter. Surprisingly, 5-10% of SEC1 was attached to a GPI anchor, but SEC2 showed no GPI anchoring. Since no differences in catalytic activity were observed among the four AChEs, and since the same percentage of GPI-AChE and TM-AChE were on the cell surface, we contend that in vivo experiments in which GPI-AChE is replaced can be interpreted solely on the basis of the altered anchoring domain.
ESTHER : Incardona_1996_Mol.Biol.Cell_7_595
PubMedSearch : Incardona_1996_Mol.Biol.Cell_7_595
PubMedID: 8730102

Title : Glycoinositol phospholipid anchor and protein C-terminus of bovine erythrocyte acetylcholinesterase: analysis by mass spectrometry and by protein and DNA sequencing - Haas_1996_Biochem.J_314_817
Author(s) : Haas R , Jackson BC , Reinhold B , Foster JD , Rosenberry TL
Ref : Biochemical Journal , 314 :817 , 1996
Abstract : Purified bovine erythrocyte acetylcholinesterase (AChE) was radiomethylated on its amine groups and incubated with bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the AChE glycoinositol phospholipid (GPI) anchor, and a C-terminal tryptic fragment that contained the residual GPI glycan was isolated by HPLC. Analysis by electrospray-ionization mass spectrometry revealed a parent ion of m/z 3798. The fragmentation patterns produced by collision-induced dissociation mass spectrometry of the +4 and +5 states of the parent ion indicated a 23-amino acid peptide in amide linkage to ethanolamine-P04-Hex-Hex-Hex(PO4-ethanolamine)(HexNAc)-Hex N(Me)2-inositol phosphate. The glycan structure is completely consistent with that obtained previously for the GPI anchor of human erythrocyte AChE except for the addition of the HexNAc substituent. A nearly complete peptide sequence was deduced from the fragmentation patterns, although four assignments were based only on single fragments of very low abundance. To resolve this uncertainty, a segment of bovine genomic DNA corresponding to the C-terminal AChE sequence was amplified by PCR. DNA sequencing established the 23-amino acid peptide sequence to be FLPKLLSATASEAPCTCSGPAHG, in agreement with the MS data and consistent with results from Edman protein sequencing. Dimerization of AChE polypeptides is mediated by intersubunit disulphide bonding in this C-terminal segment, but the bovine AChE contained two cysteine residues in a ...CTC... motif, in contrast with human AChE which contains only a single cysteine in this segment. Although bovine AChE contained no free thiol groups reactive with iodo[14C]acetamide, partial reduction and alkylation with iodo[14C]acetamide revealed that conversion into monomers occurred with an overall incorporation of only one alkyl group per monomer. An identical level of alkylation was observed when dimeric human AChE was converted into monomers by partial reduction. The question of whether the bovine AChE contains one or two intersubunit disulphide linkages is considered.
ESTHER : Haas_1996_Biochem.J_314_817
PubMedSearch : Haas_1996_Biochem.J_314_817
PubMedID: 8615775

Title : The rate of thermal inactivation of Torpedo acetylcholinesterase is not reduced in the C231S mutant - Wilson_1996_FEBS.Lett_379_161
Author(s) : Wilson EJ , Massoulie J , Bon S , Rosenberry TL
Ref : FEBS Letters , 379 :161 , 1996
Abstract : The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). At 30 degrees C or 37 degrees C, inactivation rates with 0.3 mM DTNB increased about 5-fold for the wild-type enzyme and for two site-specific mutants, D72S and V129R. The reversible active site inhibitor, ambenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, ambenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5-fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate increased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129R/C231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129R/C231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231.
ESTHER : Wilson_1996_FEBS.Lett_379_161
PubMedSearch : Wilson_1996_FEBS.Lett_379_161
PubMedID: 8635584

Title : Binding of the neurotoxin fasciculin 2 to the acetylcholinesterase peripheral site drastically reduces the association and dissociation rate constants for N-methylacridinium binding to the active site - Rosenberry_1996_Biochemistry_35_685
Author(s) : Rosenberry TL , Rabl CR , Neumann E
Ref : Biochemistry , 35 :685 , 1996
Abstract : The acetylcholinesterase (AChE) active site consists of a gorge 2 nm deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. Like certain other cationic ligands that bind specifically to the acylation site, N-methylacridinium can still interact with the acylation site in the AChE-fasciculin 2 complex. At 310 K (37 degrees C), the equilibrium dissociation constant KL' for N-methylacridinium binding to the complex was 4.0 +/- 0.7 microM, less than an order of magnitude larger than the KL = 1.0 +/- 0.3 microM for N-methylacridinium interaction with human AChE in the absence of fasciculin 2. To assess whether fasciculin 2 can sterically block access of a ligand to the acylation site, thermodynamic and kinetic constants for the interaction of N-methylacridinium with AChE in the presence and absence of fasciculin 2 were measured by fluorescence temperature jump relaxation kinetics. During progressive titration of the enzyme with increasing concentrations of N-methylacridinium, a prominent relaxation in the 0.1-1 ms range was observed in the absence of fasciculin 2. When excess fasciculin 2 was added, the prominent relaxation shifted to the 0.3-1 s range. Estimates of total AChE concentrations, KL, or KL' from analyses of relaxation amplitudes agreed well with those from equilibrium fluorescence, confirming that the relaxations corresponded to the bimolecular reactions of interest. Further analysis of the relaxation times in the absence of fasciculin 2 gave estimates of the N-methylacridinium association rate constant k12 = 8 x 10(8) M-1 s-1 and dissociation rate constant k21 = 750 s-1 at 310 K (37 degrees C). For the AChE-fasciculin 2 complex, the corresponding constants were k12' = 1.0 x 10(5) M-1 s-1 and k21' = 0.4 s-1. Thus the rate constants decreased by more than 3 orders of magnitude when fasciculin 2 was bound, consistent with a pronounced steric blockade of N-methylacridinium ingress to and egress from the acylation site.
ESTHER : Rosenberry_1996_Biochemistry_35_685
PubMedSearch : Rosenberry_1996_Biochemistry_35_685
PubMedID: 8547248

Title : Replacement of the glycoinositol phospholipid anchor of Drosophila acetylcholinesterase with a transmembrane domain does not alter sorting in neurons and epithelia but results in behavioral defects - Incardona_1996_Mol.Biol.Cell_7_613
Author(s) : Incardona JP , Rosenberry TL
Ref : Molecular Biology of the Cell , 7 :613 , 1996
Abstract : Drosophila has a single glycoinositol phospholipid (GPI)-anchored form of acetylcholinesterase (AChE) encoded by the Ace locus. To assess the role that GPI plays in the physiology, of AChE, we have replaced the wild-type GPI-AChE with a chimeric transmembrane form (TM-AChE) in the nervous system of the fly. Ace null alleles provided a genetic background completely lacking in endogenous GPI-AChE, and Ace minigene P transposon constructs were used to express both GPI- and TM-AChE forms in the tissues where AChE is normally expressed. Control experiments with the GPI-AChE minigene demonstrated a threshold between 9 and 12% of normal AChE activity for adult viability. Ace mutant flies were rescued by GPI-AChE minigene lines that expressed 12-40% of normal activity and were essentially unchanged from wild-type flies in behavior. TM-AChE minigene lines were able to rescue Ace null alleles, although with a slightly higher threshold than that for GPI-AChE. Although rescued flies expressing GPI-AChE at a level of 12% of normal activity were viable, flies expressing 13-16% of normal activity from the TM-AChE transgene died shortly after eclosion. Flies expressing TM-AChE at about 30% of normal levels were essentially unchanged from wild-type flies in gross behavior but had a reduced lifespan secondary to subtle coordination defects. These flies also showed reduced locomotor activity and performed poorly in a grooming assay. However, light level and electron microscopic immunocytochemistry showed no differences in the localization of GPI- and TM-AChE. Furthermore, endogenous and ectopic-induced expression of both AChEs in epithelial tissues of the adult and embryo, respectively, showed that they were sorted identically. Most epithelial cells sorted GPI- and TM-AChE to the apical surface, but cuticle-secreting epithelia sorted both proteins basolaterally. Our data suggest that rather than having a primary role in protein sorting, the GPI anchor or AChE plays some other more subtle cellular role in neuronal physiology.
ESTHER : Incardona_1996_Mol.Biol.Cell_7_613
PubMedSearch : Incardona_1996_Mol.Biol.Cell_7_613
PubMedID: 8730103

Title : Protein denaturation by addition and removal of acetonitrile: application to tryptic digestion of acetylcholinesterase - Haas_1995_Anal.Biochem_224_425
Author(s) : Haas R , Rosenberry TL
Ref : Analytical Biochemistry , 224 :425 , 1995
Abstract : Bovine erythrocyte acetylcholinesterase was prepared for tryptic digestion by radiomethylating with [14C]HCHO and NaCNBH3, cleaving with purified bacterial phosphatidylinositol-specific phospholipase C to remove the lipid portion of the glycoinositol phospholipid anchor, and reducing and alkylating the intersubunit disulfide bonds. Two alternative denaturation procedures were then compared prior to incubation with trypsin. In the conventional procedure, acetylcholinesterase was treated with 6 M guanidine hydrochloride for 40 min at room temperature and dialyzed. In a new procedure, acetonitrile (CH3CN) was added to 30% v/v for 10-15 min at room temperature and then removed by vacuum evaporation. The CH3CN concentration during evaporation could be estimated from the apparent pH of the solution (20 mM phosphate buffer), which varied linearly over the range of 0-75% CH3CN. CH3CN was removed in a mixture of constant composition (approximately 11% H2O-89% CH3CN), so that a final CH3CN content of 0-5% could be monitored by solution weight alone. The tryptic digests of the two denatured stocks yielded comparable HPLC profiles for A215 and radioactivity. This new denaturation protocol may be of general utility because of its convenience and gentle conditions.
ESTHER : Haas_1995_Anal.Biochem_224_425
PubMedSearch : Haas_1995_Anal.Biochem_224_425
PubMedID: 7710103

Title : Fasciculin 2 Binds to the Peripheral Site on Acetylcholinesterase and Inhibits Substrate Hydrolysis by Slowing a Step Involving Proton Transfer during Enzyme Acylation -
Author(s) : Eastman J , Wilson EJ , Cervenansky C , Rosenberry TL
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. :209 , 1995
PubMedID:

Title : Fasciculin 2 binds to the peripheral site on acetylcholinesterase and inhibits substrate hydrolysis by slowing a step involving proton transfer during enzyme acylation - Eastman_1995_J.Biol.Chem_270_19694
Author(s) : Eastman J , Wilson EJ , Cervenansky C , Rosenberry TL
Ref : Journal of Biological Chemistry , 270 :19694 , 1995
Abstract : The acetylcholinesterase active site consists of a gorge 20 A deep that is lined with aromatic residues. A serine residue near the base of the gorge defines an acylation site where an acyl enzyme intermediate is formed during the hydrolysis of ester substrates. Residues near the entrance to the gorge comprise a peripheral site where inhibitors like propidium and fasciculin 2, a snake neurotoxin, bind and interfere with catalysis. We report here the association and dissociation rate constants for fasciculin 2 interaction with the human enzyme in the presence of ligands that bind to either the peripheral site or the acylation site. These kinetic data confirmed that propidium is strictly competitive with fasciculin 2 for binding to the peripheral site. In contrast, edrophonium, N-methylacridinium, and butyrylthiocholine bound to the acylation site and formed ternary complexes with the fasciculin 2-bound enzyme in which their affinities were reduced by about an order of magnitude from their affinities in the free enzyme. Steady state analysis of the inhibition of substrate hydrolysis by fasciculin 2 revealed that the ternary complexes had residual activity. For acetylthiocholine and phenyl acetate, saturating amounts of the toxin reduced the first-order rate constant kcat to 0.5-2% and the second-order rate constant kcat/Kapp to 0.2-2% of their values with the uninhibited enzyme. To address whether fasciculin 2 inhibition primarily involved steric blockade of the active site or conformational interaction with the acylation site, deuterium oxide isotope effects on these kinetic parameters were measured. The isotope effect on kcat/Kapp increased for both substrates when fasciculin 2 was bound to the enzyme, indicating that fasciculin 2 acts predominantly by altering the conformation of the active site in the ternary complex so that steps involving proton transfer during enzyme acylation are slowed..
ESTHER : Eastman_1995_J.Biol.Chem_270_19694
PubMedSearch : Eastman_1995_J.Biol.Chem_270_19694
PubMedID: 7649979

Title : Inositol glycan phosphate derived from human erythrocyte acetylcholinesterase glycolipid anchor and inositol cyclic 1,2-phosphate antagonize glucagon activation of glycogen phosphorylase - Deeg_1993_Diabetes_42_1318
Author(s) : Deeg MA , Brass EP , Rosenberry TL
Ref : Diabetes , 42 :1318 , 1993
Abstract : In this study we examine the hypothesis that an inositol glycan phosphate can act similarly to insulin on intact cells. The inositol glycan phosphate used in this study (glycan alpha) was isolated previously from the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase and was shown to have the structure glycine-ethanolamine-PO4-Man-Man-(N,N-dimethylethanolamine-PO4)Man- (N,N-dimethyl)GlcN-inositol-PO4. The cellular response investigated was the glucagon-stimulated activation of glycogen phosphorylase in rat hepatocytes. When hepatocytes were incubated with 20 nM glucagon for 4 min, the ratio of phosphorylase a activity to total phosphorylase increased from a basal value of 0.49 +/- 0.02 to 0.82 +/- 0.03 (mean +/- SE, n = 15). Inclusion of either 100 nM insulin or 3-10 microM glycan alpha during the glucagon incubation significantly decreased the glucagon-stimulated activity ratio to 0.74 +/- 0.03 for either agent. Furthermore, hepatocyte preparations differed in their response to insulin and were divided into insulin-responsive and -resistant groups. Glycan alpha had a significant effect only in the insulin-responsive group for which the observed activity ratio for 10 microM glycan alpha plus glucagon (0.68 +/- 0.05) compared closely with that for insulin plus glucagon (0.70 +/- 0.04). For the insulin-resistant group, the activity ratio in the presence of 10 microM glycan alpha was 0.81 +/- 0.03, unchanged from the control with glucagon alone. Because glycan alpha contains an inositol phosphate group, the effect of inositol cyclic 1,2-phosphate on the glucagon-stimulated activity ratio was determined.
ESTHER : Deeg_1993_Diabetes_42_1318
PubMedSearch : Deeg_1993_Diabetes_42_1318
PubMedID: 8349043

Title : Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase. Novel fragments produced by trifluoroacetic acid - Deeg_1992_J.Biol.Chem_267_18573
Author(s) : Deeg MA , Humphrey DR , Yang SH , Ferguson TR , Reinhold VN , Rosenberry TL
Ref : Journal of Biological Chemistry , 267 :18573 , 1992
Abstract : Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted alpha-delta were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan alpha showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan beta was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans alpha and beta were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan alpha contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan beta contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50% HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.
ESTHER : Deeg_1992_J.Biol.Chem_267_18573
PubMedSearch : Deeg_1992_J.Biol.Chem_267_18573
PubMedID: 1388156

Title : Ambenonium is a rapidly reversible noncovalent inhibitor of acetylcholinesterase, with one of the highest known affinities - Hodge_1992_Mol.Pharmacol_41_937
Author(s) : Hodge AS , Humphrey DR , Rosenberry TL
Ref : Molecular Pharmacology , 41 :937 , 1992
Abstract : Steady state patterns of inhibition of purified human erythrocyte acetylcholinesterase by three inhibitors were analyzed. Edrophonium acted essentially as a competitive inhibitor, whereas tacrine and ambenonium gave mixed competitive and uncompetitive inhibition with acetylthiocholine as substrate. Inhibition constants for the competitive components were 470 microM for edrophonium, 65 microM for tacrine, and 0.12 nM for ambenonium. The extremely high affinity of ambenonium permitted analysis of the rates of approach to steady state inhibition. These rates were characterized by a single exponential time course with rate constants, kexp, that showed a linear dependence when plotted against ambenonium concentration, at fixed substrate concentration. The intercepts of these plots were independent of the substrate concentration and indicated an ambenonium dissociation rate constant of 0.013 +/- 0.002 sec-1. The slope of the plot at the lowest substrate concentration approximated the ambenonium bimolecular or association rate constant and gave a value of 5.2 +/- 0.6 x 10(7) M-1 sec-1. Three models were examined to account for the nearly linear dependence of the slopes of these plots on the substrate concentration. These models indicated that ambenonium and acetylthiocholine competed for a peripheral anionic site in the acetyl-enzyme intermediate formed during substrate hydrolysis. The apparent equilibrium dissociation constant of acetylthiocholine for this peripheral site (1.2-1.4 mM) was significantly different from that calculated from substrate inhibition data (20.1 +/- 2.8 mM). We propose that acetylthiocholine can interact with the acetyl-enzyme both at the peripheral site and at the active site but that only the latter interaction inhibits substrate hydrolysis.
ESTHER : Hodge_1992_Mol.Pharmacol_41_937
PubMedSearch : Hodge_1992_Mol.Pharmacol_41_937
PubMedID: 1588924

Title : Amphiphilic, glycophosphatidylinositol-specific phospholipase C (PI-PLC)-insensitive monomers and dimers of acetylcholinesterase - Bon_1991_Cell.Mol.Neurobiol_11_157
Author(s) : Bon S , Rosenberry TL , Massoulie J
Ref : Cellular Molecular Neurobiology , 11 :157 , 1991
Abstract : 1. In a recent study, we distinguished two classes of amphiphilic AChE3 dimers in Torpedo tissues: class I corresponds to glycolipid-anchored dimers and class II molecules are characterized by their lack of sensitivity to PI-PLC and PI-PLD, relatively small shift in sedimentation with detergent, and absence of aggregation without detergent. 2. In the present report, we analyze the amphiphlic or nonamphiphilic properties of globular AChE forms in T28 murine neural cells, rabbit muscle, and chicken muscle. The molecular forms were identified by sucrose gradient sedimentation in the presence and absence of detergent and analyzed by nondenaturing charge-shift electrophoresis. Some amphiphilic forms showed an abnormal electrophoretic migration in the absence of detergent, because of the retention of detergent micelles. 3. We show that the amphiphilic monomers (G1a) from these tissues, as well as the amphiphilic dimers (G2a) from chicken muscle, resemble the class II dimers of Torpedo AChE. We cannot exclude that these molecules possess a glycolipidic anchor but suggest that their hydrophobic domain may be of a different nature. We discuss their relationship with other cholinesterase molecular forms.
ESTHER : Bon_1991_Cell.Mol.Neurobiol_11_157
PubMedSearch : Bon_1991_Cell.Mol.Neurobiol_11_157
PubMedID: 1849452

Title : Rapid analysis of glycolipid anchors in amphiphilic dimers of acetylcholinesterases - Toutant_1991_Cell.Mol.Neurobiol_11_219
Author(s) : Toutant JP , Krall JA , Richards MK , Rosenberry TL
Ref : Cellular Molecular Neurobiology , 11 :219 , 1991
Abstract : 1. We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs). 2. Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing a diacylglycerol, but not those with an alkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Roberts et al., J. Biol. Chem. 263:18766-18775, 1988; Biochem. Biophys. Res. Commun. 150:271-277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE from Drosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols). 3. Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutant et al., Eur. J. Biochem. 180:503-508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC. 4. In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.
ESTHER : Toutant_1991_Cell.Mol.Neurobiol_11_219
PubMedSearch : Toutant_1991_Cell.Mol.Neurobiol_11_219
PubMedID: 1849455

Title : Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages - Roberts_1991_J.Biol.Chem_266_7481
Author(s) : Roberts WL , Doctor BP , Foster JD , Rosenberry TL
Ref : Journal of Biological Chemistry , 266 :7481 , 1991
Abstract : Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.
ESTHER : Roberts_1991_J.Biol.Chem_266_7481
PubMedSearch : Roberts_1991_J.Biol.Chem_266_7481
PubMedID: 2019579

Title : Amphiphilic G1 and G2 Forms of Acetylcholinesterase: Sensitivity or Resistance to Phosphatidylinositol-Specific Phospholipase C -
Author(s) : Toutant JP , Murray NR , Krall JA , Richards MK , Rosenberry TL
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :12 , 1991
PubMedID:

Title : Bovine Brain Acetylcholinesterase Sequence Involved in Intersubunit Disulfide Linkages -
Author(s) : Rosenberry TL , Roberts WL , Doctor BP
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :17 , 1991
PubMedID:

Title : Molecular forms of acetylcholinesterase in two sublines of human erythroleukemia K562 cells. Sensitivity or resistance to phosphatidylinositol-specific phospholipase C and biosynthesis - Toutant_1990_Eur.J.Biochem_187_31
Author(s) : Toutant JP , Richards MK , Krall JA , Rosenberry TL
Ref : European Journal of Biochemistry , 187 :31 , 1990
Abstract : Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N-ethylmaleimide was required to preserve the G2a form. In Triton X-100 extracts of the subline K562-243, phosphatidylinositol-specific phospholipase C (PtdIns-PLC) from Bacillus thuringiensis converted most of the G2a AChE into a hydrophilic dimer (G2h), indicating that the G2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562-243 cells with PtdIns-PLC released approximately 60% of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns-PLC was not obtained in extracts or intact cells of the subline K562-48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562-48 AChE resembles the G2a form of human erythrocyte AChE, which is resistant to PtdIns-PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns-PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503-508]. In both K562 sublines, sialidase decreased the migration of the G2a form but not of the G1a form of AChE. G1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans-Golgi apparatus. The sialidase-resistant G1a molecules were also resistant to PtdIns-PLC digestion; possible explanations for this resistance are presented.
ESTHER : Toutant_1990_Eur.J.Biochem_187_31
PubMedSearch : Toutant_1990_Eur.J.Biochem_187_31
PubMedID: 2298208

Title : Identification and analysis of glycoinositol phospholipid anchors in membrane proteins -
Author(s) : Rosenberry TL , Toutant JP , Haas R , Roberts WL
Ref : Methods Cell Biol , 32 :231 , 1989
PubMedID: 2481801

Title : Conversion of human erythrocyte acetylcholinesterase from an amphiphilic to a hydrophilic form by phosphatidylinositol-specific phospholipase C and serum phospholipase D - Toutant_1989_Eur.J.Biochem_180_503
Author(s) : Toutant JP , Roberts WL , Murray NR , Rosenberry TL
Ref : European Journal of Biochemistry , 180 :503 , 1989
Abstract : Each catalytic subunit in the amphiphilic dimer of human erythrocyte acetylcholinesterase (AChE) is anchored in the plasma membrane exclusively by a glycoinositol phospholipid. In contrast to erythrocyte AChEs in other mammalian species, the human enzyme is resistant to direct cleavage by phosphatidylinositol-specific phospholipase C (PtdIns-specific PLC). The resistance is due to the existence of an additional fatty acyl chain on the inositol ring which blocks the action of PtdIns-specific PLC [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. In this report, nondenaturing polyacrylamide gel electrophoresis was applied to permit rapid and unambiguous distinction between amphiphilic AChE, in which each catalytic subunit binds one nonionic detergent micelle, and hydrophilic AChE, which does not interact with detergent. Deacylation of human erythrocyte AChE by an alkaline treatment with hydroxylamine rendered the amphiphilic AChE susceptible to PtdIns-specific PLC with the consequent release of hydrophilic AChE. Although serum anchor-specific phospholipase D (PLD) cleaves the intact human erythrocyte AChE anchor, this treatment, as judged by nondenaturing electrophoresis, did not release hydrophilic AChE. Hydroxylamine treatment before or after PLD digestion was necessary to achieve the conversion. These observations indicate that binding of a single detergent micelle was maintained when any of the three fatty acyl or alkyl groups in the human erythrocyte AChE anchor phospholipid were retained. For proteins that can be identified following nondenaturing gel electrophoresis, these procedures provide methods both for detecting glycoinositol phospholipid anchors resistant to PtdIns-specific PLC and for indicating fatty acyl and/or alkyl chains in these anchors.
ESTHER : Toutant_1989_Eur.J.Biochem_180_503
PubMedSearch : Toutant_1989_Eur.J.Biochem_180_503
PubMedID: 2540962

Title : Lipid analysis of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase. Palmitoylation of inositol results in resistance to phosphatidylinositol-specific phospholipase C - Roberts_1988_J.Biol.Chem_263_18766
Author(s) : Roberts WL , Myher JJ , Kuksis A , Low MG , Rosenberry TL
Ref : Journal of Biological Chemistry , 263 :18766 , 1988
Abstract : The glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase (EC 3.1.1.7) contains a novel inositol phospholipid which in this and the accompanying paper (Roberts, W.L., Santikarn, S., Reinhold, V.N., and Rosenberry, T.L. (1988) J. Biol. Chem 263, 18776-18784) is shown to be a plasmanylinositol that is palmitoylated on the inositol ring. The inositol phospholipid was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I] iodophenyl)diazirine and characterized by various chemical and enzymatic cleavage procedures whose products were analyzed by thin layer chromatography and autoradiography or gas chromatography. Acidic methanolysis of human erythrocyte acetylcholinesterase (Ehu AChE) revealed 18:0 and 18:1 alkylglycerols (0.55 and 0.20 mol/mol AChE, respectively). Acetolysis was shown by TLC to release alkylacylglycerol acetates from Ehu AChE. Analysis by gas chromatography revealed that 83% of the alkylacylglycerol acetates contained an 18:0 or 18:1 1-alkyl group and a 22:4 (n - 6), 22:5 (n - 3), or 22:6 (n - 3) 2-acyl group. The inositol phospholipid is linked to the anchor by a glucosamine in glycosidic linkage, and deamination with nitrous acid cleaved the glycosidic linkage and released the phospholipid. The deamination and acetolysis products from Ehu AChE were purified by high performance liquid chromatography, and fatty acid analysis following acidic methanolysis of the purified products revealed that 2 fatty acid residues were associated with the deamination product and only one with the alkylacylglycerol acetolysis product. The other fatty acid residue was primarily palmitate and was indicated to be in ester linkage to an inositol hydroxyl(s). This linkage was shown to be responsible for the resistance of the inositol phospholipid to cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase. Deacylation of the inositol phospholipid deamination product by treatment with base removed this palmitoyl group and facilitated release of alkyl- and alkylacylglycerol species by phosphatidylinositol-specific phospholipase C with concomitant formation of inositol 1-phosphate. In contrast, digestion of Ehu AChE with a recently reported anchor-specific phospholipase D resulted in release of plasmanic acids from the intact palmitoylated plasmanylinositol.
ESTHER : Roberts_1988_J.Biol.Chem_263_18766
PubMedSearch : Roberts_1988_J.Biol.Chem_263_18766
PubMedID: 2848806

Title : Structural characterization of the glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase by fast atom bombardment mass spectrometry - Roberts_1988_J.Biol.Chem_263_18776
Author(s) : Roberts WL , Santikarn S , Reinhold VN , Rosenberry TL
Ref : Journal of Biological Chemistry , 263 :18776 , 1988
Abstract : The glycoinositol phospholipid membrane anchor of human erythrocyte acetylcholinesterase (EC 3.1.1.7) is composed of a glycan linked through a glucosamine residue to an inositol phospholipid that is resistant to the action of phosphatidylinositol-specific phospholipase C. Deamination cleavage of the glucosamine with nitrous acid released the inositol phospholipid which was purified by high performance liquid chromatography. Analysis by fast atom bombardment mass spectrometry with negative ion monitoring and by the complementary technique of collision-induced dissociation revealed molecular and daughter ions that indicated a plasmanylinositol with a palmitoyl group on an inositol hydroxyl. The intact membrane anchor was released from reductively methylated human erythrocyte acetylcholinesterase by proteolysis with papain or Pronase, deacylated by base hydrolysis, and purified by high performance liquid chromatography. Positive and negative ion fast atom bombardment mass spectrometry of the major products isolated by high performance liquid chromatography indicated the following structure for the complete glycoinositol phospholipid anchor. (formula; see text) Methylation of free amino groups by reduction with deuterium instead of hydrogen permitted determination of the number of free amino groups in individual fragment ions as further confirmation of structural assignments. The structure of the glycan portion of the human erythrocyte acetylcholinesterase membrane anchor appears to be similar to that described for Trypanosome brucei variant surface glycoprotein MITat 1.4 (variant 117) (Ferguson, M.A.J., Homans, S.W., Dwek, R.A., and Rademacher, T.W. (1988) Science 239, 753-759) except for the absence of a galactose antenna and the presence of a phosphorylethanolamine on the hexose adjacent to glucosamine.
ESTHER : Roberts_1988_J.Biol.Chem_263_18776
PubMedSearch : Roberts_1988_J.Biol.Chem_263_18776
PubMedID: 2848807

Title : Drosophila acetylcholinesterase: demonstration of a glycoinositol phospholipid anchor and an endogenous proteolytic cleavage - Haas_1988_Biochemistry_27_6453
Author(s) : Haas R , Marshall TL , Rosenberry TL
Ref : Biochemistry , 27 :6453 , 1988
Abstract : The presence of a glycoinositol phospholipid anchor in Drosophila acetylcholinesterase (AChE) was shown by several criteria. Chemical analysis of highly purified Drosophila AChE demonstrated approximately one residue of inositol per enzyme subunit. Selective cleavage by Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) was tested with Drosophila AChE radiolabeled by the photoactivatable affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID), a reagent that specifically labels the lipid moiety of glycoinositol phospholipid-anchored proteins. Digestion with PI-PLC released 75% of this radiolabel from the protein. Gel electrophoresis of Drosophila AChE in sodium dodecyl sulfate indicated prominent 55- and 16-kDa bands and a faint 70-kDa band. The [125I]TID label was localized on the 55-kDa fragment, suggesting that this fragment is the C-terminal portion of the protein. In support of this conclusion, a sensitive microsequencing procedure that involved manual Edman degradation combined with radiomethylation was used to determine residues 2-5 of the 16-kDa fragment. Comparison with the Drosophila AChE cDNA sequence [Hall, L.M.C., & Spierer, P. (1986) EMBO J. 5, 2949-2954] confirmed that the 16-kDa fragment includes the N-terminus of AChE. Furthermore, the position of the N-terminal amino acid of the mature Drosophila AChE is closely homologous to that of Torpedo AChE. The presence of radiomethylatable ethanolamine in both 16- and 55-kDa fragments was also confirmed. Thus, Drosophila AChE may include a second posttranslational modification involving ethanolamine.
ESTHER : Haas_1988_Biochemistry_27_6453
PubMedSearch : Haas_1988_Biochemistry_27_6453
PubMedID: 2975507

Title : A 13 kDa fragment is responsible for the hydrophobic aggregation of brain G4 acetylcholinesterase - Fuentes_1988_Biochem.J_256_1047
Author(s) : Fuentes ME , Rosenberry TL , Inestrosa NC
Ref : Biochemical Journal , 256 :1047 , 1988
Abstract : Proteinase K treatment of the bovine brain acetylcholinesterase (AChE) releases a hydrophobic fragment of 13 kDa, which is entirely responsible for the aggregation of the G4 AChE in the absence of detergent. This observation provides evidence that the 13 kDa fragment, which comes from a previously identified 20 kDa subunit, is directly involved in the attachment of the G4 AChE to brain membranes. A model for the organization of the different sub-domains of the hydrophobic anchor of the G4 AChE is presented.
ESTHER : Fuentes_1988_Biochem.J_256_1047
PubMedSearch : Fuentes_1988_Biochem.J_256_1047
PubMedID: 3223943

Title : Alkylacylglycerol molecular species in the glycosylinositol phospholipid membrane anchor of bovine erythrocyte acetylcholinesterase - Roberts_1988_Biochem.Biophys.Res.Commun_150_271
Author(s) : Roberts WL , Myher JJ , Kuksis A , Rosenberry TL
Ref : Biochemical & Biophysical Research Communications , 150 :271 , 1988
Abstract : Bovine erythrocyte acetylcholinesterase, a glycosylinositol phospholipid anchored membrane enzyme, was digested with phosphatidylinositol-specific phospholipase C and the released glycerol-containing moieties were identified and quantitated. About 96% of the total was alkylacylglycerol, of which sn-1-stearyl-2-stearoylglycerol, sn-1-stearyl-2-oleoylglycerol and sn-1-oleyl-2-stearoylglycerol accounted for 69%, 13% and 10%, respectively. These alkylacylglycerols are in marked contrast to the exclusively diacylglycerol species present in phosphatidylinositol from bovine erythrocyte membranes. This difference suggests that assembly of the membrane anchor of Ebo AChE involves a selected cellular pool of diradylglycerols.
ESTHER : Roberts_1988_Biochem.Biophys.Res.Commun_150_271
PubMedSearch : Roberts_1988_Biochem.Biophys.Res.Commun_150_271
PubMedID: 3337715

Title : Acetylcholinesterase from bovine caudate nucleus is attached to membranes by a novel subunit distinct from those of acetylcholinesterases in other tissues - Inestrosa_1987_J.Biol.Chem_262_4441
Author(s) : Inestrosa NC , Roberts WL , Marshall TL , Rosenberry TL
Ref : Journal of Biological Chemistry , 262 :4441 , 1987
Abstract : Acetylcholinesterase extracted with Triton X-100 from bovine brain caudate nuclei was purified by affinity chromatography to apparent homogeneity. The purified enzyme was labeled with [3H]diisopropyl fluorophosphate at the active sites and with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, a compound which has been shown to be selective for the hydrophobic membrane-binding domains of several other proteins. The subunit structure was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate before and after disulfide reduction. After reduction, a single 3H-labeled band at 70 kDa was stained by silver, but most of the 125I label corresponded to a 20-kDa species. Prior to reduction, five 3H-labeled and silver-stained bands were apparent at 70, 140, 160, 260, and greater than 360 kDa. These species were presumed to represent monomer and disulfide-linked oligomers of 70-kDa catalytic subunits. 125I label was selectively associated with the 160-, 260-, greater than 360-, and a 90-kDa species. Quantitative gel slicing of 3H- and 125I-labeled nonreduced enzyme supported a structural model in which the tetrameric enzyme is a dimer of nonidentical catalytic subunit dimers, one of which involves a direct intersubunit disulfide linkage between two 70-kDa catalytic subunit monomers and the second of which contains two disulfide linkages through an intervening 125I-labeled 20-kDa noncatalytic subunit. This 20-kDa subunit is proposed to contain the membrane attachment site. The brain enzyme did not contain components characteristic of the glycolipid anchors of erythrocyte acetylcholinesterases. However, part of the 125I label was associated with fatty acids, indicating that at least a portion of the brain enzyme membrane anchor is composed of nonamino acid components.
ESTHER : Inestrosa_1987_J.Biol.Chem_262_4441
PubMedSearch : Inestrosa_1987_J.Biol.Chem_262_4441
PubMedID: 3558347

Title : Isolation and characterization of acetylcholinesterase from Drosophila - Gnagey_1987_J.Biol.Chem_262_13290
Author(s) : Gnagey AL , Forte M , Rosenberry TL
Ref : Journal of Biological Chemistry , 262 :13290 , 1987
Abstract : The purification and characterization of acetylcholinesterase from heads of the fruit fly Drosophila are described. Sequential extraction procedures indicated that approximately 40% of the activity was soluble and 60% membrane-bound and that virtually none (less than 4%) corresponded to collagen-tailed forms. The membrane-bound enzyme was extracted with Triton X-100 and purified over 4000-fold by affinity chromatography on acridinium resin. Hydrodynamic analysis by both sucrose gradient centrifugation and chromatography on Sepharose CL-4B revealed an Mr of 165,000 similar to that observed for dimeric (G2) forms of the enzyme in mammalian tissues. In contrast, the purified enzyme gave predominant bands of about 100 kDa prior to disulfied reduction and 55 kDa after reduction on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, values that are significantly lower than those reported for purified G2 enzymes from other species. However, the presence of a faint band at 70 kDa which could be labeled by [3H]diisopropyl fluorophosphate prior to denaturation suggested that the 55-kDa band as well as a 16-kDa species arose from proteolysis. This was confirmed by reductive radiomethylation and amine analysis of the 70-, 55-, and 16-kDa bands. All three contained ethanolamine and glucosamine residues that are characteristic of a C-terminal glycolipid anchor in other G2 acetylcholinesterases. The catalytic properties of the enzyme were examined by titration with a fluorogenic reagent which revealed a turnover number for acetylthiocholine that was 6-fold lower than eel and 3-fold lower than human erythrocyte acetylcholinesterase. Furthermore, the Drosophila enzyme hydrolyzed butyrylthiocholine much more efficiently than these eel or human enzymes, an indication that the fly head enzyme has a substrate specificity intermediate between mammalian acetylcholinesterases and butyrylcholinesterases.
ESTHER : Gnagey_1987_J.Biol.Chem_262_13290
PubMedSearch : Gnagey_1987_J.Biol.Chem_262_13290
PubMedID: 3115978

Title : Differences in the glycolipid membrane anchors of bovine and human erythrocyte acetylcholinesterases - Roberts_1987_Proc.Natl.Acad.Sci.U.S.A_84_7817
Author(s) : Roberts WL , Kim BH , Rosenberry TL
Ref : Proceedings of the National Academy of Sciences of the United States of America , 84 :7817 , 1987
Abstract : Acetylcholinesterases (AcChoEases; EC 3.1.1.7) from bovine (Ebo) and human (Ehu) erythrocytes were purified to apparent homogeneity by affinity chromatography. The hydrophobic portion of the glycolipid membrane anchor of each enzyme was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Several cleavage procedures demonstrated that this radiolabel was highly selective for the fatty acid portion of the anchor in both enzymes. The labeled enzymes were digested with phosphatidylinositol (PtdIns)-specific phospholipase C (EC 3.1.4.10), and label release was assessed by polyacrylamide gel electrophoresis. About 85% of the radiolabel was cleaved from Ebo AcChoEase, whereas only 5% was released from Ehu AcChoEase. This finding agrees with a report that Ebo AcChoEase was quantitatively released from intact erythrocytes by PtdIns-specific phospholipase C but Ehu AcChoEase was not [Low, M. G. & Finean, J. B. (1977) FEBS Lett. 82, 143-146]. The two AcChoEases contained comparable amounts of the anchor components ethanolamine, glucosamine, and myo-inositol, but qualitative and quantitative differences were found in the fatty acids. Thin-layer chromatography of radiolabeled fragments generated from Ebo and Ehu AcChoEases by nitrous acid deamination revealed a major difference in the membrane anchors of the two enzymes. The fragment released from Ebo AcChoEase by this procedure comigrated with PtdIns, whereas the corresponding fragment from Ehu AcChoEase had a mobility much greater than that of PtdIns even though it contained myo-inositol and fatty acids. These studies show that 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine is useful for analysis of lipid-containing compounds and indicate that, whereas Ebo AcChoEase contains PtdIns in its glycolipid anchor, Ehu AcChoEase has a different anchor structure, which is resistant to PtdIns-specific phospholipase C. This observation suggests the existence of a class of glycolipid-anchored membrane proteins resistant to this phospholipase.
ESTHER : Roberts_1987_Proc.Natl.Acad.Sci.U.S.A_84_7817
PubMedSearch : Roberts_1987_Proc.Natl.Acad.Sci.U.S.A_84_7817
PubMedID: 3479767

Title : Brain cDNA clone for human cholinesterase - McTiernan_1987_Proc.Natl.Acad.Sci.U.S.A_84_6682
Author(s) : McTiernan C , Adkins S , Chatonnet A , Vaughan TA , Bartels CF , Kott M , Rosenberry TL , La Du BN , Lockridge O
Ref : Proceedings of the National Academy of Sciences of the United States of America , 84 :6682 , 1987
Abstract : A cDNA library from human basal ganglia was screened with oligonucleotide probes corresponding to portions of the amino acid sequence of human serum cholinesterase (EC 3.1.1.8). Five overlapping clones, representing 2.4 kilobases, were isolated. The sequenced cDNA contained 207 base pairs of coding sequence 5' to the amino terminus of the mature protein in which there were four ATG translation start sites in the same reading frame as the protein. Only the ATG coding for Met-(-28) lay within a favorable consensus sequence for functional initiators. There were 1722 base pairs of coding sequence corresponding to the protein found circulating in human serum. The amino acid sequence deduced from the cDNA exactly matched the 574 amino acid sequence of human serum cholinesterase, as previously determined by Edman degradation. Therefore, our clones represented cholinesterase (EC 3.1.1.8) rather than acetylcholinesterase (EC 3.1.1.7). It was concluded that the amino acid sequences of cholinesterase from two different tissues, human brain and human serum, were identical. Hybridization of genomic DNA blots suggested that a single gene, or very few genes, coded for cholinesterase.
ESTHER : McTiernan_1987_Proc.Natl.Acad.Sci.U.S.A_84_6682
PubMedSearch : McTiernan_1987_Proc.Natl.Acad.Sci.U.S.A_84_6682
PubMedID: 3477799
Gene_locus related to this paper: human-BCHE

Title : Glycolipid membrane-binding domain of human erythrocyte acetylcholinesterase. - Rosenberry_1986_Fed.Proc_45_2970
Author(s) : Rosenberry TL , Roberts WL , Haas R
Ref : Federation Proceedings , 45 :2970 , 1986
Abstract : The membrane-binding domain of human erythrocyte acetylcholinesterase is a small hydrophobic structure at the COOH-terminus of the enzyme subunits. Papain digestion cleaves a COOH-terminal dipeptide linked to the hydrophobic structure with the sequence His-Gly-ethanolamine-Z, where the ethanolamine is in amide linkage to the glycine and Z is a partially characterized glycolipid. This glycolipid includes a second residue of ethanolamine and a residue of glucosamine, both of which have free primary amino groups accessible to radiomethylation. The glycolipid also contains a carbohydrate residue or residues that bind to concanavalin A and nearly stoichiometric amounts of both palmitate and C22 unsaturated fatty acids. Similarities in this membrane-binding structure to those reported for trypanosome variant surface glycoproteins and Thy-1 glycoprotein suggest an important new category of posttranslational modifications involving the attachment of COOH-terminal glycolipid.
ESTHER : Rosenberry_1986_Fed.Proc_45_2970
PubMedSearch : Rosenberry_1986_Fed.Proc_45_2970
PubMedID: 3536597

Title : Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase - Roberts_1986_Biochemistry_25_3091
Author(s) : Roberts WL , Rosenberry TL
Ref : Biochemistry , 25 :3091 , 1986
Abstract : The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.
ESTHER : Roberts_1986_Biochemistry_25_3091
PubMedSearch : Roberts_1986_Biochemistry_25_3091
PubMedID: 3524670

Title : Identification of amine components in a glycolipid membrane-binding domain at the C-terminus of human erythrocyte acetylcholinesterase - Haas_1986_Biochemistry_25_3098
Author(s) : Haas R , Brandt PT , Knight J , Rosenberry TL
Ref : Biochemistry , 25 :3098 , 1986
Abstract : Purified human erythrocyte acetylcholinesterase was labeled by reductive radiomethylation with saturating amounts of [14C]formaldehyde and sodium cyanoborohydride. Acid hydrolysis and automated amino acid analysis permitted both identification of radiomethylated components by their coelution with radiomethylated standards and quantitation of these components. The methylated N-terminal amino acids glutamate and arginine were observed at levels of 0.66 and 0.34 residues, respectively, per 70-kilodalton subunit, and lysine residues were methylated on their epsilon-amino groups to a level of 7.40 residues per subunit [Haas, R., & Rosenberry, T.L. (1985) Anal. Biochem. 148, 154-162]. In addition, each subunit contained 1.35 residues of methylated ethanolamine and 0.98 residue of methylated glucosamine. Papain digestion cleaved the intact enzyme into two fragments, an enzymatically active hydrophilic fragment and a small hydrophobic fragment that represented the membrane-binding domain. The radiomethylated amino acids were quantitatively retained in the hydrophilic fragment, while the methylated ethanolamine and glucosamine were confined exclusively to the hydrophobic domain fragment. This fragment included the C-terminal dipeptide of the subunit. Peptide sequencing by manual Edman methods was combined with radiomethylation to demonstrate the sequence His-Gly-ethanolamine-Z for the hydrophobic domain fragment. The ethanolamine residue in this sequence is in amide linkage to the C-terminal Gly and is clearly distinct from the ethanolamine residues in Z which are susceptible to radiomethylation in the intact enzyme. Since Z also includes glucosamine and 2 mol of fatty acids [Roberts, W.L. & Rosenberry, T.L. (1985) Biochem. Biophys. Res. Commun. 133, 621-627], we conclude that the membrane-binding domain of human erythrocyte acetylcholinesterase is a covalently linked glycolipid at the C-termini of the subunits.
ESTHER : Haas_1986_Biochemistry_25_3098
PubMedSearch : Haas_1986_Biochemistry_25_3098
PubMedID: 3524671

Title : Ionic strength dependence of the inhibition of acetylcholinesterase activity by Al3+ - Sharp_1985_Biophys.Chem_21_261
Author(s) : Sharp TR , Rosenberry TL
Ref : Biophysical Chemistry , 21 :261 , 1985
Abstract : Inhibition of acetylcholinesterase activity by Al3+ has been examined by initial velocity kinetics and by a first-order kinetic method. Both methods yield an inhibition constant of approx. 1.7 mM at 0.1 M ionic strength. The initial velocity study indicates a noncompetitive mechanism of inhibition by Al3+. Inhibition at 10 mM ionic strength shows a Ki of 0.03 mM. Evaluation of the ionic strength dependence concurs with the results of Nolte et al. (Biochemistry 19 (1980) 3705). An effective charge in the binding site of -9 predicts the ratio of inhibition constants at high and low ionic strength. Extrapolation to zero ionic strength gives a Ki0 = 0.34 microM.
ESTHER : Sharp_1985_Biophys.Chem_21_261
PubMedSearch : Sharp_1985_Biophys.Chem_21_261
PubMedID: 3986284

Title : Quantitative identification of N-terminal amino acids in proteins by radiolabeled reductive methylation and amino acid analysis: application to human erythrocyte acetylcholinesterase - Haas_1985_Anal.Biochem_148_154
Author(s) : Haas R , Rosenberry TL
Ref : Analytical Biochemistry , 148 :154 , 1985
Abstract : A novel method of determining N-terminal amino acids in proteins is introduced. Reductive methylation of a protein with radiolabeled formaldehyde methylates both the alpha-amino group of the N-terminal amino acid and the epsilon-amino groups of Lys residues. The radiomethylated amino acids are stable to acid hydrolysis, and each of 16 possible hydrolysis-stable N-terminal amino acids can be identified by the unique elution positions of its N alpha-methyl and N alpha,N alpha-dimethyl derivatives with an appropriate amino acid analyzer elution schedule. The technique is at least as sensitive as other N-terminal amino acid determinations and, in addition, permits a quantitative evaluation of the number of N-terminal groups in a sample. Reductive methylation of bovine serum albumin revealed N-terminal Asp at a stoichiometry of 0.97 amino acid residue per polypeptide, while methylation of prolactin resulted in 0.86 residue of N-terminal Thr per polypeptide. Human erythrocyte acetylcholinesterase contained two N-terminal amino acids with stoichiometries of 0.66 Glu and 0.34 Arg per 70-kDa subunit. Identification of Glu as the principal N-terminus of acetylcholinesterase was confirmed by Edman sequencing.
ESTHER : Haas_1985_Anal.Biochem_148_154
PubMedSearch : Haas_1985_Anal.Biochem_148_154
PubMedID: 4037298

Title : A small hydrophobic domain that localizes human erythrocyte acetylcholinesterase in liposomal membranes is cleaved by papain digestion - Kim_1985_Biochemistry_24_3586
Author(s) : Kim BH , Rosenberry TL
Ref : Biochemistry , 24 :3586 , 1985
Abstract : A small hydrophobic domain in isolated human erythrocyte acetylcholinesterase is responsible for the interaction of this enzyme with detergent micelles and the aggregation of the enzyme on removal of detergent. Papain has been shown to cleave this hydrophobic domain and to generate a fully active hydrophilic enzyme that shows no tendency to interact with detergents or to aggregate [Dutta-Choudhury, T.A., & Rosenberry, T.L. (1984) J. Biol. Chem. 259, 5653-5660]. We report here that the intact enzyme could be reconstituted into phospholipid liposomes while the papain-disaggregated enzyme showed no capacity for reconstitution. More than 80% of the enzyme reconstituted into small liposomes could be released by papain digestion as the hydrophilic form. Papain was less effective in releasing the enzyme from large liposomes that were probably multilamellar. In a novel application of affinity chromatography on acridinium resin, enzyme reconstituted into small liposomes in the presence of excess phospholipid was purified to a level of 1 enzyme molecule per 4000 phospholipid molecules, a ratio expected if each enzyme molecule was associated with a small, unilamellar liposome. Subunits in the hydrophilic enzyme form released from reconstituted liposomes by papain digestion showed a mass decrease of about 2 kilodaltons relative to the intact subunits according to acrylamide gel electrophoresis in sodium dodecyl sulfate, a difference similar to that observed previously following papain digestion of the soluble enzyme aggregates. The data were consistent with the hypothesis that the same hydrophobic domain in the enzyme is responsible for the interaction of the enzyme with detergent micelles, the aggregation of the enzyme in the absence of detergent, and the incorporation of the enzyme into reconstituted phospholipid membranes.
ESTHER : Kim_1985_Biochemistry_24_3586
PubMedSearch : Kim_1985_Biochemistry_24_3586
PubMedID: 4041429

Title : Identification of covalently attached fatty acids in the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase - Roberts_1985_Biochem.Biophys.Res.Commun_133_621
Author(s) : Roberts WL , Rosenberry TL
Ref : Biochemical & Biophysical Research Communications , 133 :621 , 1985
Abstract : Human erythrocyte acetylcholinesterase is an amphipathic enzyme whose hydrophobic membrane-binding domain can be selectively labeled with a lipophilic photoreagent and removed by digestion with papain. In this paper we demonstrate that methanolysis releases covalently bound fatty acids from the hydrophobic domain and thus confirm that this domain is a covalently linked glycolipid at the enzyme subunit C-terminus. About one mole of saturated and one mole of unsaturated fatty acids were released per mole of domain. Since the predominant unsaturated fatty acids (22:4 and 22:5) are minor components of the esterified fatty acid pool in human erythrocyte membranes, assembly of the glycolipid must involve a selected unsaturated fatty acid pool.
ESTHER : Roberts_1985_Biochem.Biophys.Res.Commun_133_621
PubMedSearch : Roberts_1985_Biochem.Biophys.Res.Commun_133_621
PubMedID: 4084290

Title : Human erythrocyte acetylcholinesterase is an amphipatic form -
Author(s) : Rosenberry TL , Scoggin DM , Dutta-Choudhury TA , Haas R
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter :155 , 1984
PubMedID:

Title : Structure of human erythrocyte acetylcholinesterase. Characterization of intersubunit disulfide bonding and detergent interaction - Rosenberry_1984_J.Biol.Chem_259_5643
Author(s) : Rosenberry TL , Scoggin DM
Ref : Journal of Biological Chemistry , 259 :5643 , 1984
Abstract : A large scale purification procedure for human erythrocyte acetylcholinesterase that involved affinity chromatography on an acridinium resin permitted the routine isolation of about 5 mg of enzyme from 10 liters of outdated erythrocytes. The purified enzyme had a specific activity of 5000-5800 units/mg of protein and was free of polypeptide contaminants by gel electrophoresis criteria. In detergents, the isolated enzyme corresponded to a disulfide-linked dimer (G2) that was converted to 75-kDa subunit monomers (G1) by reduction with dithiothreitol. No free sulfhydryl groups were detected prior to reduction, but reduction under nondenaturing conditions generated active G1 and produced 1.7 mol of free sulfhydryl groups/mol of subunit. These data were interpreted as indicating a single intersubunit disulfide bond in the G2 enzyme. In the absence of nonionic detergents, both the G2 and the G1 enzymes formed aggregates with average Stokes radii of 10 nm. Introduction of Triton X-100 gave enzyme-detergent complexes according to hydrodynamic criteria. Quantitative determination of [3H]Triton X-100 binding to G2 and G1 by a novel affinity chromatography procedure revealed that each G2 molecule bound about 140 detergent molecules and each G1, about 80. These observations indicated that each subunit in both G2 and G1 interacted individually with a Triton X-100 micelle. Molecular weight estimates for the protein components of the G2- and G1-detergent complexes were obtained from the hydrodynamic properties and the detergent binding data and corresponded to 160,000 and 85,000, respectively. Data in this and the accompanying paper (Dutta-Choudhury, T.A., and Rosenberry, T. L. (1984) J. Biol. Chem. 259, 5653-5660) provide strong evidence that erythrocyte acetylcholinesterase is an amphipathic protein.
ESTHER : Rosenberry_1984_J.Biol.Chem_259_5643
PubMedSearch : Rosenberry_1984_J.Biol.Chem_259_5643
PubMedID: 6715363

Title : Human erythrocyte acetylcholinesterase is an amphipathic protein whose short membrane-binding domain is removed by papain digestion - Dutta-Choudhury_1984_J.Biol.Chem_259_5653
Author(s) : Dutta-Choudhury TA , Rosenberry TL
Ref : Journal of Biological Chemistry , 259 :5653 , 1984
Abstract : Human erythrocyte acetylcholinesterase was shown to be an amphipathic protein in which proteases could cleave the hydrophobic domain from the enzymatically active hydrophilic domain. Papain and Pronase cleaved these domains with greatest efficiency, as measured by the disaggregation of purified acetylcholinesterase to disulfide-linked dimers (G2) on sucrose density gradients in the absence of detergent. Nonspecific proteolytic degradation was reduced both by the inclusion of edrophonium chloride, which protected acetylcholinesterase from inactivation, and by covalent attachment of papain to Sepharose CL-4B. In contrast to nondigested control acetylcholinesterase, the papain-disaggregated enzyme did not bind detergent according to hydrodynamic criteria and could not be reconstituted into liposomes. Thus, we conclude that the hydrophobic domain removed by papain digestion is in fact the membrane-binding domain in situ. This domain appeared largely inaccessible to proteases in intact erythrocytes, however, as less than 10% of the enzyme activity was solubilized by protease digestion. The hydrophobic domain removed by papain appeared very small, as nondigested control and disaggregated enzyme were identical in molecular weight and amino acid composition within experimental error. The fully reduced 75-kDa catalytic subunits of nondigested control enzyme appeared about 2 kDa larger than the corresponding subunits of disaggregated enzyme on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, an indication that the hydrophobic domain was cleaved from the COOH or NH2 terminus of the catalytic subunit primary structure. Studies in which the NH-terminal amino acid was labeled by reductive methylation suggested that the hydrophobic domain is at the COOH terminus.
ESTHER : Dutta-Choudhury_1984_J.Biol.Chem_259_5653
PubMedSearch : Dutta-Choudhury_1984_J.Biol.Chem_259_5653
PubMedID: 6371009

Title : Acetylcholinesterase of human erythrocytes and neuromuscular junctions: homologies revealed by monoclonal antibodies - Fambrough_1982_Proc.Natl.Acad.Sci.U.S.A_79_1078
Author(s) : Fambrough DM , Engel AG , Rosenberry TL
Ref : Proceedings of the National Academy of Sciences of the United States of America , 79 :1078 , 1982
Abstract : Human erythrocyte acetylcholinesterase was used to immunize mice, and hybridomas were generated by fusion of mouse spleen cells with cells of the Sp 2/0 myeloma cell line. Five independently derived hybridoma clones produced antibodies that bound to purified erythrocyte acetylcholinesterase. All of these antibodies crossreacted with human and monkey neuromuscular junctions; immunocytochemical staining patterns corresponded to the distribution of junctional acetylcholinesterase. The monoclonal antibodies fell into at least four categories based on differences in crossreactivity with neuromuscular acetylcholinesterase of rabbit, dog, calf, and guinea pig, and competition tests indicated that the antibodies defined five different antigenic sites on the acetylcholinesterase molecule. It is concluded that there is a high level of homology between the acetylcholinesterases of erythrocytes and neuromuscular junctions.
ESTHER : Fambrough_1982_Proc.Natl.Acad.Sci.U.S.A_79_1078
PubMedSearch : Fambrough_1982_Proc.Natl.Acad.Sci.U.S.A_79_1078
PubMedID: 6175961

Title : Acetylcholinesterase -
Author(s) : Rosenberry TL , Barnett P , Mays C
Ref : Methods Enzymol , 82 :325 , 1982
PubMedID: 6804757

Title : A pseudo-first-order kinetic approach to measurement of acetylcholine hydrolysis by acetylcholinesterase - Sharp_1982_J.Biochem.Biophys.Meth_6_159
Author(s) : Sharp TR , Rosenberry TL
Ref : Journal of Biochemical & Biophysical Methods , 6 :159 , 1982
Abstract : A continuous spectrophotometric procedure is presented for the measurement of the kinetic properties of acetylcholinesterase (EC 3.1.1.7) with its natural substrate, acetylcholine. The procedure is based upon the production of stoichiometric quantities of H+ upon hydrolysis of substrate. The spectrophotometric reporter is the pH indicator dye, phenol red and the procedure yields continuous time courses for hydrolysis of substrate. Further, this phenol red system and an adaptation of the Ellman et al. (1961, Biochem. Pharmacol. 7, 88-95) procedure for acetylthiocholine as substrate, are described as a rapid screening technique for reversible competitive and noncompetitive inhibitors of acetylcholinesterase activity. The methods are illustrated by determinations of KI for edrophonium, decamethonium and Al3+.
ESTHER : Sharp_1982_J.Biochem.Biophys.Meth_6_159
PubMedSearch : Sharp_1982_J.Biochem.Biophys.Meth_6_159
PubMedID: 7108127

Title : Cellular localization of the molecular forms of acetylcholinesterase in rat diaphragm -
Author(s) : Younkin SG , Rosenstein C , Collins PL , Rosenberry TL
Ref : Journal of Biological Chemistry , 257 :13630 , 1982
PubMedID: 7142169

Title : Characterization of pepsin-resistant collagen-like tail subunit fragments of 18S and 14S acetylcholinesterase from Electrophorus electricus - Mays_1981_Biochemistry_20_2810
Author(s) : Mays C , Rosenberry TL
Ref : Biochemistry , 20 :2810 , 1981
Abstract : Digestion of 18S and 14S acetylcholinesterase from eel electric organ with pepsin at 15 degrees C for 6 h results in extensive degradation of the catalytic subunits, but a major portion of the collagen-like tail structure associated with these enzyme forms resists degradation. The pepsin-resistant structures partially aggregate and can be isolated by gel exclusion chromatography on Sepharose CL-6B in buffered 1 M sodium chloride. The largest structure, denoted F3, has a molecular weight of 72 000 according to gel electrophoresis in sodium dodecyl sulfate and is composed of three 24 000 molecular weight polypeptides linked by intersubunit disulfide bonds. This structure is largely, but not completely, a collagen-like triple helix as indicated by a circular dichroism spectrum typical of triple-helical collagen and an amino acid composition characterized by 27% glycine, 5% hydroxyproline, and 5% hydroxylysine. Continued pepsin action results in degradation of the disulfide linkage region such that disulfide-linked dimers F2 and finally F1 monomers become the predominant forms in sodium dodecyl sulfate. Digested samples in which either F3 or F2 predominate have virtually identical circular dichroic spectra and amino acid compositions and generate similar diffuse 24 000 molecular weight polypeptides following disulfide reduction. Thus the intersubunit disulfide linkages in F3 must occur close to the end(s) of the fragment polypeptide chains. Pepsin conversion of F3 to F2 is particularly accelerated between 25 and 30 degrees C, suggesting that the triple-helical structure in the disulfide linkage region undergoes thermal destabilization in this temperature range. Digestion at 40 degrees C yields presumably triple-helical F1 structures devoid of disulfide linkages, although their degradation to small fragments can be detected at this temperature. The question of whether the three tail subunits that give rise to F1 polypeptides are identical remains open.
ESTHER : Mays_1981_Biochemistry_20_2810
PubMedSearch : Mays_1981_Biochemistry_20_2810
PubMedID: 6788073

Title : Microcomputer interface for computer-assisted enzyme kinetic studies with UV--VIS spectrophotometers -
Author(s) : Sharp TR , Gopinath KR , Brandt PW , Rosenberry TL
Ref : Analytical Biochemistry , 116 :545 , 1981
PubMedID: 7316182

Title : Effective charge on acetylcholinesterase active sites determined from the ionic strength dependence of association rate constants with cationic ligands - Nolte_1980_Biochemistry_19_3705
Author(s) : Nolte HJ , Rosenberry TL , Neumann E
Ref : Biochemistry , 19 :3705 , 1980
Abstract : The reaction of the specific fluorescent cationic ligand N-methylacridinium with the active site of 11S acetylcholinesterase from electric eel was monitored by temperature-jump relaxation kinetics at a variety of ionic strengths. The ionic strength dependence of the bimolecular association rate constant is analyzed with a Brnsted-Debye-Hckel expression and leads to estimates of the association rate constant at zero ionic strength of K120 = 1.1 X 10(10) M-1 S-1 at 25 degrees C and the net charge number of the enzyme active site of ZE = -6.3. The ionic strength dependence of the second-order hydrolysis rate constant kcat/Kapp for acetylthiocholine under steady-state conditions is also very pronounced and indicates a value of ZE = -9. Thus, a large effective negative charge on the enzyme active site appears to be a general characteristic of its interaction with cationic ligands. The ionic strength dependence of Kcat/Kapp is identical with that of sodium chloride, sodium phosphate, and sodium citrate, thus ruling out any possibility that the phenomena arise from a specific, partially competitive binding of Na+ to the enzyme active site. Substitution of the calculated electrostatic parameters into theoretical equations indicates that the most significant effect of these ZE values is a 2-3 order of magnitude reduction in the rate constant for dissociation of the initial ligand-enzyme encounter complex; this decrease renders the bimolecular reaction diffusion controlled. The high value of k120 and the space requirements of six to nine charged groups suggest that regions of the enzyme surface area larger than the catalytic sites themselves are effective in trapping cationic ligands.
ESTHER : Nolte_1980_Biochemistry_19_3705
PubMedSearch : Nolte_1980_Biochemistry_19_3705
PubMedID: 7407068

Title : Functional identity of catalytic subunits of acetylcholinesterase - Barnett_1979_Biochim.Biophys.Acta_567_154
Author(s) : Barnett P , Rosenberry TL
Ref : Biochimica & Biophysica Acta , 567 :154 , 1979
Abstract : 11 S acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from the electric eel Electrophorus electricus essentially consists of four catalytic subunits which appear to be identical structurally but to be assembled with slight asymmetry. During isolation and storage of the enzyme, proteolysis cleaves a portion of the subunits into major fragments containing the active site and minor fragments containing no active sites without change in the enzyme molecular weight. A previous report (Gentinetta, R. and Brodbeck, U. (1976) Biochim. Biophys. Acta 438 437--448) indicated that the intact and the fragmented subunits reacted with diisopropylfluorophosphate at different rates and that the reaction rate in the presence of excess phosphorylating agent was not strictly first order. Those findings could not be reproduced in this report. Intact and fragmented subunits were observed to react at the same rate with diisopropylfluorophosphate. In addition, the overall reaction kinetics both of 11 S and 18 S plus 14 S acetylcholinesterase were found to be strictly first order in the presence of an excess of diisopropylfluorophosphate throughout the course of reaction. These results are consistent with several previous reports that only one type of active site can be detected in acetylcholinesterase. The proteolysis which fragments a portion of the catalytic subunit has no apparent effect on the catalytic properties of the enzyme.
ESTHER : Barnett_1979_Biochim.Biophys.Acta_567_154
PubMedSearch : Barnett_1979_Biochim.Biophys.Acta_567_154
PubMedID: 454619

Title : Electrophysiological studies of thymectomized and nonthymectomized acetylcholine receptor-immunized animal models of myasthenia gravis -
Author(s) : Niemi WD , Nastuk WL , Chang HW , Penn AS , Rosenberry TL
Ref : Experimental Neurology , 63 :1 , 1979
PubMedID: 223859

Title : Quantitative simulation of endplate currents at neuromuscular junctions based on the reaction of acetylcholine with acetylcholine receptor and acetylcholinesterase - Rosenberry_1979_Biophys.J_26_263
Author(s) : Rosenberry TL
Ref : Biophysical Journal , 26 :263 , 1979
Abstract : Two kinetic models are introduced which predict amplitudes and time-courses of endplate currents and miniature endplate currents at neuromuscular junctions, at both normal and acetylcholinesterase-inhibited endplates. Appropriate differential rate equations reflecting interactions of acetylcholine with acetylcholine receptor and with esterase, diffusion of acetylcholine both within and from the synaptic cleft, and cooperativity between receptor site occupancy and ion channel opening are solved. Acetylcholine release into the cleft is assumed to be instantaneous. The simpler homogeneous reaction space model accurately predicts decay phase time constants are inaccurate. The two-reaction space model predicts amplitudes and time constants within a factor of two of those observed experimentally. The simulations indicate that the amplitudes and time-courses are primarily determined by the chemical reaction rates that characterize acetylcholine interactions with receptor and esterase and that these interactions occur under nonequilibrium conditions. Approximately 50% of the total ion channels in the initial reaction space are predicted to be opened at the peak endplate current. The cooperative opening of ion channels by acetylcholine requires that acetylcholine be introduced into the cleft in discrete, concentrated elements. Virtually all the open channels are confined to the initial reaction space, although acetylcholine-bound receptor sites can be much more widely distributed.
ESTHER : Rosenberry_1979_Biophys.J_26_263
PubMedSearch : Rosenberry_1979_Biophys.J_26_263
PubMedID: 262418

Title : Inactivation of Electrophorus electricus acetylcholinesterase by benzenemethane sulfonylfluoride -
Author(s) : Barnett P , Rosenberry TL
Ref : Archives of Biochemistry & Biophysics , 190 :202 , 1978
PubMedID: 708070

Title : Structure of 18S and 14S acetylcholinesterase. Identification of collagen-like subunits that are linked by disulfide bonds to catalytic subunits -
Author(s) : Rosenberry TL , Richardson JM
Ref : Biochemistry , 16 :3550 , 1977
PubMedID: 889810

Title : Interaction of ligands with acetylcholinesterase. Use of temperature-jump relaxation kinetics in the binding of specific fluorescent ligands -
Author(s) : Rosenberry TL , Neumann E
Ref : Biochemistry , 16 :3870 , 1977
PubMedID: 20130

Title : Catalysis by acetylcholinesterase. Acceleration of the hydrolysis of neutral acetic acid esters by certain aromatic cations -
Author(s) : Barnett P , Rosenberry TL
Ref : Journal of Biological Chemistry , 252 :7200 , 1977
PubMedID: 903357

Title : Identification of discrete disulfide-linked oligomers which distinguish 18 S from 14 S acetylcholinesterase -
Author(s) : McCann WF , Rosenberry TL
Ref : Archives of Biochemistry & Biophysics , 183 :347 , 1977
PubMedID: 562134

Title : A membrane activation cycle induced by sulfhydryl reagents after affinity labeling of the acetylcholine receptor of electroplax -
Author(s) : Bartels-Bernal E , Rosenberry TL , Chang HW
Ref : Molecular Pharmacology , 12 :813 , 1976
PubMedID: 995129

Title : Catalysis by acetylcholinesterase: evidence that the rate-limiting step for acylation with certain substrates precedes general acid-base catalysis - Rosenberry_1975_Proc.Natl.Acad.Sci.U.S.A_72_3834
Author(s) : Rosenberry TL
Ref : Proceedings of the National Academy of Sciences of the United States of America , 72 :3834 , 1975
Abstract : Inferences about the catalytic mechanism of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) are frequently made on the basis of a presumed analogy with chymotrypsin, EC 3.4.21.1. Although both enzymes are serine hydrolases, several differences in the steady-state kinetic properties of the two have been observed. In this report particular attention is focused on the second-order reaction constant, kcat/Kapp. While the reported pH dependence and deuterium oxide isotope effect associated with this parameter for chymotrypsin are generally consistent with simple models involving rate-limiting general acid-base catalysis, this study finds a more complicated situation with acetylcholinesterase. The apparent pKa of kcat/Kapp for acetylcholinesterase varies between 5.5 and 6.3 for neutral substrates and involves nonlinear inhibition by [H+]. Deuterium oxide isotope effects for kcat/Kapp range from 1.1 for acetylcholine to 1.9 for p-nitrophenyl acetate. The bimolecular reaction rate appears rate-limiting for acetylcholine at low concentrations, while a rate-limiting induced-fit step is proposed to account for apparent pKa values and low deuterium oxide isotope effects associated with low concentrations of phenyl acetate and isoamyl acetate.
ESTHER : Rosenberry_1975_Proc.Natl.Acad.Sci.U.S.A_72_3834
PubMedSearch : Rosenberry_1975_Proc.Natl.Acad.Sci.U.S.A_72_3834
PubMedID: 668

Title : Acetylcholinesterase. - Rosenberry_1975_Adv.Enzymol.Relat.Areas.Mol.Biol_43_103
Author(s) : Rosenberry TL
Ref : Adv Enzymol Relat Areas Mol Biol , 43 :103 , 1975
Abstract :
ESTHER : Rosenberry_1975_Adv.Enzymol.Relat.Areas.Mol.Biol_43_103
PubMedSearch : Rosenberry_1975_Adv.Enzymol.Relat.Areas.Mol.Biol_43_103
PubMedID: 891

Title : Catalysis by acetylcholinesterase. The rate-limiting steps involved in theacylation of acetylcholinesterase by acetic acid esters and phosphorylating agents -
Author(s) : Rosenberry TL
Ref : Croatica Chemica Acta , 47 :235 , 1975
PubMedID:

Title : General discussion on models for the ligand binding sites of cholinesterase -
Author(s) : Reiner E , Aldridge WN , Hopff WH , Taylor P , Krupka RM , Rosenberry TL , Augustinsson KB , Mooser G , O'Brien RD , Brodbeck U , Massoulie J , Maricic S , Eldefrawi ME , Silman I
Ref : Croatica Chemica Acta , 47 :499 , 1975
PubMedID:

Title : Subunit heterogeneity of acetylcholinesterase -
Author(s) : Chen YT , Rosenberry TL , Chang HW
Ref : Archives of Biochemistry & Biophysics , 161 :479 , 1974
PubMedID: 4839042

Title : Structure of 11S acetylcholinesterase. Subunit composition -
Author(s) : Rosenberry TL , Chen YT , Bock E
Ref : Biochemistry , 13 :3068 , 1974
PubMedID: 4841055

Title : Purification of acetylcholinesterase by affinity chromatography and determination of active site stoichiometry -
Author(s) : Rosenberry TL , Chang HW , Chen YT
Ref : Journal of Biological Chemistry , 247 :1555 , 1972
PubMedID: 5012324

Title : Studies of catalysis by acetylcholinesterase. Synergistic effects of inhibitors during the hydrolysis of acetic acid esters -
Author(s) : Rosenberry TL , Bernhard SA
Ref : Biochemistry , 11 :4308 , 1972
PubMedID: 5079901

Title : Studies of catalysis by acetylcholinesterase. I. Fluorescent titration with a carbamoylating agent -
Author(s) : Rosenberry TL , Bernhard SA
Ref : Biochemistry , 10 :4114 , 1971
PubMedID: 5168614