Silman I

General

Full name : Silman Israel

First name : Israel

Mail : Department of Neurobiology\; Weizmann Institute of Science\; Rehovot 76100

Zip Code : 76100

City : Rehovot

Country : Israel

Email : Israel.Silman@weizmann.ac.il

Phone : +972-8-9343669

Fax : +972-8-947-1949

Website : \/\/www.weizmann.ac.il\/neurobiology\/labs\/silman\/silman.html

Directory :

References (266)

Title : Do 'Newly Born' orphan proteins resemble 'Never Born' proteins? A study using three deep learning algorithms - Liu_2023_Proteins__
Author(s) : Liu J , Yuan R , Shao W , Wang J , Silman I , Sussman JL
Ref : Proteins , : , 2023
Abstract : "Newly Born" proteins, devoid of detectable homology to any other proteins, known as orphan proteins, occur in a single species or within a taxonomically restricted gene family. They are generated by the expression of novel open reading frames, and appear throughout evolution. We were curious if three recently developed programs for predicting protein structures, namely, AlphaFold2, RoseTTAFold, and ESMFold, might be of value for comparison of such "Newly Born" proteins to random polypeptides with amino acid content similar to that of native proteins, which have been called "Never Born" proteins. The programs were used to compare the structures of two sets of "Never Born" proteins that had been expressed-Group 1, which had been shown experimentally to possess substantial secondary structure, and Group 3, which had been shown to be intrinsically disordered. Overall, although the models generated were scored as being of low quality, they nevertheless revealed some general principles. Specifically, all four members of Group 1 were predicted to be compact by all three algorithms, in agreement with the experimental data, whereas the members of Group 3 were predicted to be very extended, as would be expected for intrinsically disordered proteins, again consistent with the experimental data. These predicted differences were shown to be statistically significant by comparing their accessible surface areas. The three programs were then used to predict the structures of three orphan proteins whose crystal structures had been solved, two of which display novel folds. Surprisingly, only for the protein which did not have a novel fold, and was taxonomically restricted, rather than being a true orphan, did all three algorithms predict very similar, high-quality structures, closely resembling the crystal structure. Finally, they were used to predict the structures of seven orphan proteins with well-identified biological functions, whose 3D structures are not known. Two proteins, which were predicted to be disordered based on their sequences, are predicted by all three structure algorithms to be extended structures. The other five were predicted to be compact structures with only two exceptions in the case of AlphaFold2. All three prediction algorithms make remarkably similar and high-quality predictions for one large protein, HCO_11565, from a nematode. It is conjectured that this is due to many homologs in the taxonomically restricted family of which it is a member, and to the fact that the Dali server revealed several nonrelated proteins with similar folds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:Proteins:3.
ESTHER : Liu_2023_Proteins__
PubMedSearch : Liu_2023_Proteins__
PubMedID: 37092778

Title : Discovery of a Potent Dual Inhibitor of Acetylcholinesterase and Butyrylcholinesterase with Antioxidant Activity that Alleviates Alzheimer-like Pathology in Old APP\/PS1 Mice - Viayna_2021_J.Med.Chem_64_812
Author(s) : Viayna E , Coquelle N , Cieslikiewicz-Bouet M , Cisternas P , Oliva CA , Sanchez-Lopez E , Ettcheto M , Bartolini M , De Simone A , Ricchini M , Rendina M , Pons M , Firuzi O , Perez B , Saso L , Andrisano V , Nachon F , Brazzolotto X , Garcia ML , Camins A , Silman I , Jean L , Inestrosa NC , Colletier JP , Renard PY , Munoz-Torrero D
Ref : Journal of Medicinal Chemistry , 64 :812 , 2021
Abstract : The combination of the scaffolds of the cholinesterase inhibitor huprine Y and the antioxidant capsaicin results in compounds with nanomolar potencies toward human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) that retain or improve the antioxidant properties of capsaicin. Crystal structures of their complexes with AChE and BChE revealed the molecular basis for their high potency. Brain penetration was confirmed by biodistribution studies in C57BL6 mice, with one compound (5i) displaying better brain/plasma ratio than donepezil. Chronic treatment of 10 month-old APP/PS1 mice with 5i (2 mg/kg, i.p., 3 times per week, 4 weeks) rescued learning and memory impairments, as measured by three different behavioral tests, delayed the Alzheimer-like pathology progression, as suggested by a significantly reduced Abeta42/Abeta40 ratio in the hippocampus, improved basal synaptic efficacy, and significantly reduced hippocampal oxidative stress and neuroinflammation. Compound 5i emerges as an interesting anti-Alzheimer lead with beneficial effects on cognitive symptoms and on some underlying disease mechanisms.
ESTHER : Viayna_2021_J.Med.Chem_64_812
PubMedSearch : Viayna_2021_J.Med.Chem_64_812
PubMedID: 33356266
Gene_locus related to this paper: human-ACHE

Title : Torpedo californica acetylcholinesterase is stabilized by binding of a divalent metal ion to a novel and versatile 4D motif - Silman_2021_Protein.Sci_30_966
Author(s) : Silman I , Shnyrov VL , Ashani Y , Roth E , Nicolas A , Sussman JL , Weiner L
Ref : Protein Science , 30 :966 , 2021
Abstract : Stabilization of Torpedo californica acetylcholinesterase by the divalent cations Ca(+2) , Mg(+2) and Mn(+2) was investigated. All three substantially protect the enzyme from thermal inactivation. Electron paramagnetic resonance revealed one high-affinity binding site for Mn(+2) and several much weaker sites. Differential scanning calorimetry showed a single irreversible thermal transition. All three cations raise both the temperature of the transition and the activation energy, with the transition becoming more cooperative. The crystal structures of the Ca(+2) and Mg(+2) complexes with Torpedo acetylcholinesterase were solved. A principal binding site was identified. In both cases, it consists of four aspartates (a 4D motif), within which the divalent ion is embedded, together with several waters molecules. It makes direct contact with two of the aspartates, and indirect contact, via waters, with the other two. The 4D motif has been identified in 31 acetylcholinesterase sequences and 28 butyrylcholinesterase sequences. Zebrafish acetylcholinesterase also contains the 4D motif; it, too, is stabilized by divalent metal ions. The ASSAM server retrieved 200 other proteins that display the 4D motif, in many of which it is occupied by a divalent cation. It is a very versatile motif, since, even though tightly conserved in terms of rmsd values, it can contain from one to as many as three divalent metal ions, together with a variable number of waters. This novel motif, which binds primarily divalent metal ions, is shared by a broad repertoire of proteins. This article is protected by copyright. All rights reserved.
ESTHER : Silman_2021_Protein.Sci_30_966
PubMedSearch : Silman_2021_Protein.Sci_30_966
PubMedID: 33686648
Gene_locus related to this paper: torca-ACHE

Title : The multiple biological roles of the cholinesterases - Silman_2021_Prog.Biophys.Mol.Biol_162_41
Author(s) : Silman I
Ref : Prog Biophys Mol Biol , 162 :41 , 2021
Abstract : It is tacitly assumed that the biological role of acetylcholinesterase is termination of synaptic transmission at cholinergic synapses. However, together with its structural homolog, butyrylcholinesterase, it is widely distributed both within and outside the nervous system, and, in many cases, the role of both enzymes remains obscure. The transient appearance of the cholinesterases in embryonic tissues is especially enigmatic. The two enzymes' extra-synaptic roles, which are known as 'non-classical' roles, are the topic of this review. Strong evidence has been presented that AChE and BChE play morphogenetic roles in a variety of eukaryotic systems, and they do so either by acting as adhesion proteins, or as trophic factors. As trophic factors, one mode of action is to directly regulate morphogenesis, such as neurite outgrowth, by poorly understood mechanisms. The other mode is by regulating levels of acetylcholine, which acts as the direct trophic factor. Alternate substrates have been sought for the cholinesterases. Quite recently, it was shown that levels of the aggression hormone, ghrelin, which also controls appetite, are regulated by butyrylcholinesterase. The rapid hydrolysis of acetylcholine by acetylcholinesterase generates high local proton concentrations. The possible biophysical and biological consequences of this effect are discussed. The biological significance of the acetylcholinesterases secreted by parasitic nematodes is reviewed, and, finally, the involvement of acetylcholinesterase in apoptosis is considered.
ESTHER : Silman_2021_Prog.Biophys.Mol.Biol_162_41
PubMedSearch : Silman_2021_Prog.Biophys.Mol.Biol_162_41
PubMedID: 33307019

Title : Preface: Cholinergic mechanisms: This is the Preface for the special issue Cholinergic Mechanisms - Anglister_2021_J.Neurochem__
Author(s) : Anglister L , Silman I , Soreq H
Ref : Journal of Neurochemistry , : , 2021
Abstract : This special issue of the Journal of Neurochemistry, entitled "Cholinergic Mechanisms," presents 15 reviews and two original papers, which have been selected to cover the broad spectrum of topics and disciplines presented at the XVIth International Symposium on Cholinergic Mechanisms (ISCM-XVI), ranging from the molecular and the cellular to the clinical and the cognitive mechanisms of cholinergic transmission. The authors discuss recent developments in the field, for instance, the association of cholinergic transmission with a number of important neurological and neuromuscular diseases in the central and peripheral nervous systems.
ESTHER : Anglister_2021_J.Neurochem__
PubMedSearch : Anglister_2021_J.Neurochem__
PubMedID: 34458988

Title : Computational studies on cholinesterases: Strengthening our understanding of the integration of structure, dynamics and function - Sussman_2020_Neuropharmacol__108265
Author(s) : Sussman JL , Silman I
Ref : Neuropharmacology , :108265 , 2020
Abstract : Computational approaches have proved valuable in elucidating structure/function relationships in the cholinesterases in the context of their unusual three-dimensional structure. In this review we survey several recent studies that have enhanced our understanding of how these enzymes function, and have utilized computational approaches both to modulate their activity and to improve the design of lead compounds for their inhibition.
ESTHER : Sussman_2020_Neuropharmacol__108265
PubMedSearch : Sussman_2020_Neuropharmacol__108265
PubMedID: 32795461

Title : Polyproline-rich peptides associated with Torpedo californica acetylcholinesterase tetramers - Toker_2020_Chem.Biol.Interact__109007
Author(s) : Toker L , Silman I , Zeev-Ben-Mordehai T , Sussman JL , Schopfer LM , Lockridge O
Ref : Chemico-Biological Interactions , :109007 , 2020
Abstract : Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the polyproline-rich peptide(s) in Torpedo californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo californica electric organ, converted to 300kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the polyproline-rich peptide in the trypsin-treated TcAChE tetramers.
ESTHER : Toker_2020_Chem.Biol.Interact__109007
PubMedSearch : Toker_2020_Chem.Biol.Interact__109007
PubMedID: 32087110

Title : Chiral Separation, X-ray Structure, and Biological Evaluation of a Potent and Reversible Dual Binding Site AChE Inhibitor - Catto_2020_ACS.Med.Chem.Lett_11_869
Author(s) : Catto M , Pisani L , De la Mora E , Belviso BD , Mangiatordi GF , Pinto A , Palma A , Denora N , Caliandro R , Colletier JP , Silman I , Nicolotti O , Altomare CD
Ref : ACS Med Chem Lett , 11 :869 , 2020
Abstract : Acetylcholinesterase (AChE) inhibitors (AChEIs) still remain the leading therapeutic options for the symptomatic treatment of cognitive deficits associated with mild-to-moderate Alzheimer's disease. The search for new AChEIs benefits from well-established knowledge of the molecular interactions of selective AChEIs, such as donepezil and related dual binding site inhibitors. Starting from a previously disclosed coumarin-based inhibitor (+/-)-cis-1, active as racemate in the nanomolar range toward AChE, we proceeded on a double track by (i) achieving chiral resolution of the enantiomers of 1 by HPLC and (ii) preparing two close achiral analogues of 1, i.e., compounds 4 and 6. An eudismic ratio as high as 20 was observed for the (-) enantiomer of cis-1. The X-ray crystal structure of the complex between the (-)-cis-1 eutomer (coded as MC1420) and T. californica AChE was determined at 2.8 A, and docking calculation results suggested that the eutomer in (1R,3S) absolute configuration should be energetically more favored in binding the enzyme than the eutomer in (1S,3R) configuration. The achiral analogues 4 and 6 were less effective in inhibiting AChE compared to (+/-)-cis-1, but interestingly butylamide 4 emerged as a potent inhibitor of butyrylcholinesterase (BChE).
ESTHER : Catto_2020_ACS.Med.Chem.Lett_11_869
PubMedSearch : Catto_2020_ACS.Med.Chem.Lett_11_869
PubMedID: 32435398

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 : Rivastigmine and metabolite analogues with putative Alzheimer's disease-modifying properties in a Caenorhabditis elegans model. - Dighe_2019_Commun.Chem_2_35
Author(s) : Dighe SN , De la Mora E , Chan S , Kantham S , McColl G , Miles JA , Veliyath SK , Sreenivas BY , Nassar ZD , Silman I , Sussman JL , Weik M , McGeary RP , Parat MO , Brazzolotto X , Ross BP
Ref : Communications chemistry , 2 :35 , 2019
Abstract : The development of polyphenols as drugs for Alzheimer's disease (AD) is thwarted by their meagre brain availability due to instability and poor druglikeness. Here we describe the successful development of stable, druglike polyphenolic analogues of the current AD drug rivastigmine, that have high apparent blood-brain barrier permeabilities and multifunctional properties for AD treatment. The compounds inhibit cholinesterases and amyloid beta (Abeta) fibrillation, protect against Abeta42-induced toxicity in vitro, and demonstrate efficacy in vivo in a transgenic Caenorhabditis elegans model expressing Abeta42, with potencies similar to rivastigmine and natural polyphenols. The results suggest that a tertiary amine substituent is amenable for developing water-soluble, membrane-permeable polyphenols, and its incorporation adjacent to a hydroxy group is favourable for intramolecular hydrogen bonding that facilitates membrane permeability. Carbamylation of one hydroxy group protects the polyphenols from degradation and mostly improves their membrane permeability. These design strategies may assist in the development of polyphenol-based drugs.
ESTHER : Dighe_2019_Commun.Chem_2_35
PubMedSearch : Dighe_2019_Commun.Chem_2_35
PubMedID:
Gene_locus related to this paper: human-BCHE , torca-ACHE

Title : Molecular dynamics simulations of the interaction of Mouse and Torpedo acetylcholinesterase with covalent inhibitors explain their differential reactivity: Implications for drug design - Chandar_2019_Chem.Biol.Interact_13ChEPon_310_108715
Author(s) : Chandar NB , Efremenko I , Silman I , Martin JML , Sussman JL
Ref : Chemico-Biological Interactions , 310 :108715 , 2019
Abstract : Although the three-dimensional structures of mouse and Torpedo californica acetylcholinesterase are very similar, their responses to the covalent sulfonylating agents benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride are qualitatively different. Both agents inhibit the mouse enzyme effectively by covalent modification of its active-site serine. In contrast, whereas the Torpedo enzyme is effectively inhibited by benzenesulfonyl fluoride, it is almost completely resistant to phenylmethylsulfonyl fluoride. A bottleneck midway down the active-site gorge in both enzymes restricts access of ligands to the active site at the bottom of the gorge. Molecular dynamics simulations revealed that the mouse enzyme is substantially more flexible than the Torpedo enzyme, suggesting that enhanced 'breathing motions' of the mouse enzyme relative to the Torpedo enzyme may explain why phenylmethylsulfonyl fluoride can reach the active site in mouse acetylcholinesterase, but not in the Torpedo enzyme. Accordingly, we performed docking of the two sulfonylating agents to the two enzymes, followed by molecular dynamics simulations. Whereas benzenesulfonyl fluoride closely approaches the active-site serine in both mouse and Torpedo acetylcholinesterase in such simulations, phenylmethylsulfonyl fluoride is able to approach the active-site serine of mouse acetylcholinesterase, but remains trapped above the bottleneck in the Torpedo enzyme. Our studies demonstrate that reliance on docking tools in drug design can produce misleading information. Docking studies should, therefore, also be complemented by molecular dynamics simulations in selection of lead compounds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:2.
ESTHER : Chandar_2019_Chem.Biol.Interact_13ChEPon_310_108715
PubMedSearch : Chandar_2019_Chem.Biol.Interact_13ChEPon_310_108715
PubMedID: 31226285

Title : Novel multitarget-directed ligands targeting acetylcholinesterase and sigma1 receptors as lead compounds for treatment of Alzheimer's disease: Synthesis, evaluation, and structural characterization of their complexes with acetylcholinesterase - Lalut_2019_Eur.J.Med.Chem_162_234
Author(s) : Lalut J , Santoni G , Karila D , Lecoutey C , Davis A , Nachon F , Silman I , Sussman JL , Weik M , Maurice T , Dallemagne P , Rochais C
Ref : Eur Journal of Medicinal Chemistry , 162 :234 , 2019
Abstract : Pleiotropic intervention may be a requirement for effective limitation of the progression of multifactorial diseases such as Alzheimer's Disease. One approach to such intervention is to design a single chemical entity capable of acting on two or more targets of interest, which are accordingly known as Multi-Target Directed Ligands (MTDLs). We recently described donecopride, the first MTDL able to simultaneously inhibit acetylcholinesterase and act as an agonist of the 5-HT4 receptor, which displays promising activities in vivo. Pharmacomodulation of donecopride allowed us to develop a novel series of indole derivatives possessing interesting in vitro activities toward AChE and the sigma1 receptor. The crystal structures of complexes of the most promising compounds with Torpedo californica AChE were solved in order to further understand their mode of inhibition.
ESTHER : Lalut_2019_Eur.J.Med.Chem_162_234
PubMedSearch : Lalut_2019_Eur.J.Med.Chem_162_234
PubMedID: 30447434
Gene_locus related to this paper: torca-ACHE

Title : Design, biological evaluation and X-ray crystallography of nanomolar multifunctional ligands targeting simultaneously acetylcholinesterase and glycogen synthase kinase-3 - Oukoloff_2019_Eur.J.Med.Chem_168_58
Author(s) : Oukoloff K , Coquelle N , Bartolini M , Naldi M , Le Guevel R , Bach S , Josselin B , Ruchaud S , Catto M , Pisani L , Denora N , Iacobazzi RM , Silman I , Sussman JL , Buron F , Colletier JP , Jean L , Routier S , Renard PY
Ref : Eur Journal of Medicinal Chemistry , 168 :58 , 2019
Abstract : Both cholinesterases (AChE and BChE) and kinases, such as GSK-3alpha/beta, are associated with the pathology of Alzheimer's disease. Two scaffolds, targeting AChE (tacrine) and GSK-3alpha/beta (valmerin) simultaneously, were assembled, using copper(I)-catalysed azide alkyne cycloaddition (CuAAC), to generate a new series of multifunctional ligands. A series of eight multi-target directed ligands (MTDLs) was synthesized and evaluated in vitro and in cell cultures. Molecular docking studies, together with the crystal structures of three MTDL/TcAChE complexes, with three tacrine-valmerin hybrids allowed designing an appropriate linker containing a 1,2,3-triazole moiety whose incorporation preserved, and even increased, the original inhibitory potencies of the two selected pharmacophores toward the two targets. Most of the new derivatives exhibited nanomolar affinity for both targets, and the most potent compound of the series displayed inhibitory potencies of 9.5nM for human acetylcholinesterase (hAChE) and 7nM for GSK-3alpha/beta. These novel dual MTDLs may serve as suitable leads for further development, since, in the micromolar range, they exhibited low cytotoxicity on a panel of representative human cell lines including the human neuroblastoma cell line SH-SY5Y. Moreover, these tacrine-valmerin hybrids displayed a good ability to penetrate the blood-brain barrier (BBB) without interacting with efflux pumps such as P-gp.
ESTHER : Oukoloff_2019_Eur.J.Med.Chem_168_58
PubMedSearch : Oukoloff_2019_Eur.J.Med.Chem_168_58
PubMedID: 30798053
Gene_locus related to this paper: torca-ACHE

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

Title : Increasing Polarity in Tacrine and Huprine Derivatives: Potent Anticholinesterase Agents for the Treatment of Myasthenia Gravis - Galdeano_2018_Molecules_23_
Author(s) : Galdeano C , Coquelle N , Cieslikiewicz-Bouet M , Bartolini M , Perez B , Clos MV , Silman I , Jean L , Colletier JP , Renard PY , Munoz-Torrero D
Ref : Molecules , 23 : , 2018
Abstract : Symptomatic treatment of myasthenia gravis is based on the use of peripherally-acting acetylcholinesterase (AChE) inhibitors that, in some cases, must be discontinued due to the occurrence of a number of side-effects. Thus, new AChE inhibitors are being developed and investigated for their potential use against this disease. Here, we have explored two alternative approaches to get access to peripherally-acting AChE inhibitors as new agents against myasthenia gravis, by structural modification of the brain permeable anti-Alzheimer AChE inhibitors tacrine, 6-chlorotacrine, and huprine Y. Both quaternization upon methylation of the quinoline nitrogen atom, and tethering of a triazole ring, with, in some cases, the additional incorporation of a polyphenol-like moiety, result in more polar compounds with higher inhibitory activity against human AChE (up to 190-fold) and butyrylcholinesterase (up to 40-fold) than pyridostigmine, the standard drug for symptomatic treatment of myasthenia gravis. The novel compounds are furthermore devoid of brain permeability, thereby emerging as interesting leads against myasthenia gravis.
ESTHER : Galdeano_2018_Molecules_23_
PubMedSearch : Galdeano_2018_Molecules_23_
PubMedID: 29534488
Gene_locus related to this paper: torca-ACHE

Title : Cryo-EM structure of the native butyrylcholinesterase tetramer reveals a dimer of dimers stabilized by a superhelical assembly - Leung_2018_Proc.Natl.Acad.Sci.U.S.A_115_13270
Author(s) : Leung MR , van Bezouwen LS , Schopfer LM , Sussman JL , Silman I , Lockridge O , Zeev-Ben-Mordehai T
Ref : Proc Natl Acad Sci U S A , 115 :13270 , 2018
Abstract : The quaternary structures of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are essential for their localization and function. Of practical importance, BChE is a promising therapeutic candidate for intoxication by organophosphate nerve agents and insecticides, and for detoxification of addictive substances. Efficacy of the recombinant enzyme hinges on its having a long circulatory half-life; this, in turn, depends strongly on its ability to tetramerize. Here, we used cryoelectron microscopy (cryo-EM) to determine the structure of the highly glycosylated native BChE tetramer purified from human plasma at 5.7 A. Our structure reveals that the BChE tetramer is organized as a staggered dimer of dimers. Tetramerization is mediated by assembly of the C-terminal tryptophan amphiphilic tetramerization (WAT) helices from each subunit as a superhelical assembly around a central lamellipodin-derived oligopeptide with a proline-rich attachment domain (PRAD) sequence that adopts a polyproline II helical conformation and runs antiparallel. The catalytic domains within a dimer are asymmetrically linked to the WAT/PRAD. In the resulting arrangement, the tetramerization domain is largely shielded by the catalytic domains, which may contribute to the stability of the human BChE (HuBChE) tetramer. Our cryo-EM structure reveals the basis for assembly of the native tetramers and has implications for the therapeutic applications of HuBChE. This mode of tetramerization is seen only in the cholinesterases but may provide a promising template for designing other proteins with improved circulatory residence times.
ESTHER : Leung_2018_Proc.Natl.Acad.Sci.U.S.A_115_13270
PubMedSearch : Leung_2018_Proc.Natl.Acad.Sci.U.S.A_115_13270
PubMedID: 30538207
Gene_locus related to this paper: human-BCHE

Title : Structure-Based Optimization of Nonquaternary Reactivators of Acetylcholinesterase Inhibited by Organophosphorus Nerve Agents - Santoni_2018_J.Med.Chem_61_7630
Author(s) : Santoni G , de Sousa J , De la Mora E , Dias J , Jean L , Sussman JL , Silman I , Renard PY , Brown RCD , Weik M , Baati R , Nachon F
Ref : Journal of Medicinal Chemistry , 61 :7630 , 2018
Abstract : Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE. X-ray structures and molecular docking indicate that structural modification of the tetrahydroacridine might decrease inhibition without affecting reactivation. The chlorinated derivative was synthesized and, in line with the prediction, displayed a 10-fold decrease in inhibition but no significant decrease in reactivation efficiency. X-ray structures with the derivative rationalize this outcome. We thus show that rational design based on structural studies permits the refinement of new-generation pyridine aldoxime reactivators that may be more effective in the treatment of nerve agent intoxication.
ESTHER : Santoni_2018_J.Med.Chem_61_7630
PubMedSearch : Santoni_2018_J.Med.Chem_61_7630
PubMedID: 30125110
Gene_locus related to this paper: torca-ACHE

Title : Potent 3-Hydroxy-2-Pyridine Aldoxime Reactivators of Organophosphate-Inhibited Cholinesterases with Predicted Blood-Brain Barrier Penetration - Zorbaz_2018_Chemistry_24_9675
Author(s) : Zorbaz T , Braiki A , Marakovic N , Renou J , De la Mora E , Macek Hrvat N , Katalinic M , Silman I , Sussman JL , Mercey G , Gomez C , Mougeot R , Perez B , Baati R , Nachon F , Weik M , Jean L , Kovarik Z , Renard PY
Ref : Chemistry , 24 :9675 , 2018
Abstract : A new series of 3-hydroxy-2-pyridine aldoxime compounds have been designed, synthesised and tested in vitro, in silico, and ex vivo as reactivators of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) inhibited by organophosphates (OPs), for example, VX, sarin, cyclosarin, tabun, and paraoxon. The reactivation rates of three oximes (16-18) were determined to be greater than that of 2-PAM and comparable to that of HI-6, two pyridinium aldoximes currently used by the armies of several countries. The interactions important for a productive orientation of the oxime group within the OP-inhibited enzyme have been clarified by molecular-modelling studies, and by the resolution of the crystal structure of the complex of oxime 17 with Torpedo californica AChE. Blood-brain barrier penetration was predicted for oximes 15-18 based on their physicochemical properties and an in vitro brain membrane permeation assay. Among the evaluated compounds, two morpholine-3-hydroxypyridine aldoxime conjugates proved to be promising reactivators of OP-inhibited cholinesterases. Moreover, efficient ex vivo reactivation of phosphylated native cholinesterases by selected oximes enabled significant hydrolysis of VX, sarin, paraoxon, and cyclosarin in whole human blood, which indicates that the oximes have scavenging potential.
ESTHER : Zorbaz_2018_Chemistry_24_9675
PubMedSearch : Zorbaz_2018_Chemistry_24_9675
PubMedID: 29672968
Gene_locus related to this paper: torca-ACHE

Title : Generation of Reactive Oxygen Species by Photosensitizers and their Modes of Action on Proteins - Bilkis_2018_Curr.Med.Chem_25_5528
Author(s) : Bilkis I , Silman I , Weiner L
Ref : Curr Med Chem , 25 :5528 , 2018
Abstract : In this review, we first survey the mechanisms underlying the chemical modification of amino acid residues in proteins by singlet oxygen elicited by photosensitizers. Singlet oxygen has the capacity to cause widespread chemical damage to cellular proteins. Its use in photodynamic therapy of tumors thus requires the development of methodologies for specific addressing of the photosensitizer to malignant cells while sparing normal tissue. We describe three targeting paradigms for achieving this objective. The first involves the use of a photosensitizer with a high affinity for its target protein; in this case, the photosensitizer is methylene blue for acetylcholinesterase. The second paradigm involves the use of the hydrophobic photosensitizer hypericin, which has the capacity to interact selectively with partially unfolded forms of proteins, including nascent species in rapidly dividing or virus-infected and cancer cells, acting preferentially at membrane interfaces. In this case, partially unfolded molten globule species of acetylcholinesterase serve as the model system. In the third paradigm, the photodynamic approach takes advantage of a general approach in 'state-of-the-art' chemotherapy, by coupling the photosensitizer emodin to a specific peptide hormone, GnRH, which recognizes malignant cells via specific GnRH receptors on their surface.
ESTHER : Bilkis_2018_Curr.Med.Chem_25_5528
PubMedSearch : Bilkis_2018_Curr.Med.Chem_25_5528
PubMedID: 29303072

Title : Preface: Cholinergic Mechanisms - Prado_2017_J.Neurochem_142 Suppl 2_3
Author(s) : Prado MAM , Marchot P , Silman I
Ref : Journal of Neurochemistry , 142 Suppl 2 :3 , 2017
Abstract : This special issue is a companion to the meeting 'XVth International Symposium on Cholinergic Mechanisms', and is edited by Israel Silman, Marco Prado and Pascale Marchot. In the review articles, renowned researchers in the field capture key mechanisms of cholinergic neurotransmission, from genomic amplification of cholinesterase genes, splicing and post-translational modifications; features of the neuromuscular junction, implications of cholinergic circuitry that are relevant to addiction, anxiety and mood, to preclinical models, protein biomarkers, and clinical findings that are relevant to pathology, for example, developmental neurotoxicity. The broad variety of features reflects the impact of cholinergic mechanisms on many physiological events and emphasizes the importance of research in this area. This is the Preface for the special issue XVth International Symposium on Cholinergic Mechanisms.
ESTHER : Prado_2017_J.Neurochem_142 Suppl 2_3
PubMedSearch : Prado_2017_J.Neurochem_142 Suppl 2_3
PubMedID: 28791707

Title : Recent developments in structural studies on acetylcholinesterase - Silman_2017_J.Neurochem_142 Suppl 2_19
Author(s) : Silman I , Sussman JL
Ref : Journal of Neurochemistry , 142 Suppl 2 :19 , 2017
Abstract : This review focuses on several recent developments concerning structure-function relationships in vertebrate acetylcholinesterase. These include studies on high-resolution structures of human acetylcholinesterase and its complexes; the first crystal structure of a snake venom acetylcholinesterase, in which open and closed states of the 'back door' are visualized; a powerful algorithm for redesigning proteins for enhanced expression in prokaryotic systems, as applied to human acetylcholinesterase, which has hitherto been an intractable target; in situ implementation of 'click chemistry' in crystalline acetylcholinesterase, which yields novel insights into the steric and dynamic changes involved in the reaction within the active-site gorge; and a study that demonstrates the effect of crystallization conditions on ligand alignment within a protein complex, in this case the methylene blue-Torpedo californica acetylcholinesterase complex, which highlights the relevance of the precipitant employed to structure-based drug design. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.
ESTHER : Silman_2017_J.Neurochem_142 Suppl 2_19
PubMedSearch : Silman_2017_J.Neurochem_142 Suppl 2_19
PubMedID: 28503857

Title : Collagen XIII secures pre- and postsynaptic integrity of the neuromuscular synapse - Haronen_2017_Hum.Mol.Genet_26_2076
Author(s) : Haronen H , Zainul Z , Tu H , Naumenko N , Sormunen R , Miinalainen I , Shakirzyanova A , Oikarainen T , Abdullin A , Martin P , Santoleri S , Koistinaho J , Silman I , Giniatullin R , Fox MA , Heikkinen A , Pihlajaniemi T
Ref : Hum Mol Genet , 26 :2076 , 2017
Abstract : Both transmembrane and extracellular cues, one of which is collagen XIII, regulate the formation and function of the neuromuscular synapse, and their absence results in myasthenia. We show that the phenotypical changes in collagen XIII knock-out mice are milder than symptoms in human patients, but the Col13a1-/- mice recapitulate major muscle findings of congenital myasthenic syndrome type 19 and serve as a disease model. In the lack of collagen XIII neuromuscular synapses do not reach full size, alignment, complexity and function resulting in reduced muscle strength. Collagen XIII is particularly important for the preterminal integrity, and when absent, destabilization of the motor nerves results in muscle regeneration and in atrophy especially in the case of slow muscle fibers. Collagen XIII was found to affect synaptic integrity through binding the ColQ tail of acetylcholine esterase. Although collagen XIII is a muscle-bound transmembrane molecule, it also undergoes ectodomain shedding to become a synaptic basal lamina component. We investigated the two forms' roles by novel Col13a1tm/tm mice in which ectodomain shedding is impaired. While postsynaptic maturation, terminal branching and neurotransmission was exaggerated in the Col13a1tm/tm mice, the transmembrane form's presence sufficed to prevent defects in transsynaptic adhesion, Schwann cell invagination/retraction, vesicle accumulation and acetylcholine receptor clustering and acetylcholinesterase dispersion seen in the Col13a1-/- mice, pointing to the transmembrane form as the major conductor of collagen XIII effects. Altogether, collagen XIII secures postsynaptic, synaptic and presynaptic integrity, and it is required for gaining and maintaining normal size, complexity and functional capacity of the neuromuscular synapse.
ESTHER : Haronen_2017_Hum.Mol.Genet_26_2076
PubMedSearch : Haronen_2017_Hum.Mol.Genet_26_2076
PubMedID: 28369367

Title : Computational Studies on Acetylcholinesterases - Xu_2017_Molecules_22_
Author(s) : Xu Y , Cheng S , Sussman JL , Silman I , Jiang H
Ref : Molecules , 22 : , 2017
Abstract : Functions of biomolecules, in particular enzymes, are usually modulated by structural fluctuations. This is especially the case in a gated diffusion-controlled reaction catalyzed by an enzyme such as acetylcholinesterase. The catalytic triad of acetylcholinesterase is located at the bottom of a long and narrow gorge, but it catalyzes the extremely rapid hydrolysis of the neurotransmitter, acetylcholine, with a reaction rate close to the diffusion-controlled limit. Computational modeling and simulation have produced considerable advances in exploring the dynamical and conformational properties of biomolecules, not only aiding in interpreting the experimental data, but also providing insights into the internal motions of the biomolecule at the atomic level. Given the remarkably high catalytic efficiency and the importance of acetylcholinesterase in drug development, great efforts have been made to understand the dynamics associated with its functions by use of various computational methods. Here, we present a comprehensive overview of recent computational studies on acetylcholinesterase, expanding our views of the enzyme from a microstate of a single structure to conformational ensembles, strengthening our understanding of the integration of structure, dynamics and function associated with the enzyme, and promoting the structure-based and/or mechanism-based design of new inhibitors for it.
ESTHER : Xu_2017_Molecules_22_
PubMedSearch : Xu_2017_Molecules_22_
PubMedID: 28796192

Title : Alexander George Karczmar (1917-2017) - Soreq_2017_J.Neurochem_143_609
Author(s) : Soreq H , Silman I
Ref : Journal of Neurochemistry , 143 :609 , 2017
Abstract : The neurochemistry community at large and the Advisory Board of The International Symposia on Cholinergic Mechanisms mourn the loss of Alexander George Karczmar, the elected Honorary President of these international symposia, who passed away peacefully in his Chicago home at the age of 100 on August 17, 2017. For many of us Alex was the essence of cholinergic signaling, and personified its versatile power to send messages between the brain and the peripheral tissues and organs, and to connect between body and soul.
ESTHER : Soreq_2017_J.Neurochem_143_609
PubMedSearch : Soreq_2017_J.Neurochem_143_609
PubMedID: 29027666

Title : In vitro evaluation of the catalytic activity of paraoxonases and phosphotriesterases predicts the enzyme circulatory levels required for in vivo protection against organophosphate intoxications - Ashani_2016_Chem.Biol.Interact_259_252
Author(s) : Ashani Y , Leader H , Aggarwal N , Silman I , Worek F , Sussman JL , Goldsmith M
Ref : Chemico-Biological Interactions , 259 :252 , 2016
Abstract : Catalytic scavengers of organophosphates (OPs) are considered very promising antidote candidates for preventing the adverse effects of OP intoxication as stand alone treatments. This study aimed at correlating the in-vivo catalytic efficiency ((kcat/KM)[Enzyme]pl), established prior to the OP challenge, with the severity of symptoms and survival rates of intoxicated animals. The major objective was to apply a theoretical approach to estimate a lower limit for (kcat/KM)[Enzyme]pl that will be adequate for establishing the desired kcat/KM value and plasma concentration of efficacious catalytic bioscavengers. Published data sets by our group and others, from in vivo protection experiments executed in the absence of any supportive medicine, were analyzed. The kcat/KM values of eight OP hydrolyzing enzymes and their plasma concentrations in four species exposed to OPs via s.c., i.m. and oral gavage, were analyzed. Our results show that regardless of the OP type and the animal species employed, sign-free animals were observed following bioscavenger treatment provided the theoretically estimated time period required to detoxify 96% of the OP (t96%) in-vivo was </=10 s. This, for example, can be achieved by an enzyme with kcat/KM = 5 x 107 M-1 min-1 and a plasma concentration of 0.4 muM ((kcat/KM)[Enzyme]pl = 20 min-1). Experiments in which animals were intoxicated by i.v. OP injections did not always conform to this rule, and in some cases resulted in high mortality rates. We suggest that in vivo evaluation of catalytic scavengers should avoid the unrealistic bolus i.v. route of OP exposure.
ESTHER : Ashani_2016_Chem.Biol.Interact_259_252
PubMedSearch : Ashani_2016_Chem.Biol.Interact_259_252
PubMedID: 27163850

Title : The Impact of Crystallization Conditions on Structure-Based Drug Design: A Case Study on the Methylene Blue\/Acetylcholinesterase Complex - Dym_2016_Protein.Sci_25_1096
Author(s) : Dym O , Song W , Felder CE , Roth E , Shnyrov V , Ashani Y , Xu Y , Joosten RP , Weiner L , Sussman JL , Silman I
Ref : Protein Science , 25 :1096 , 2016
Abstract : Structure-based drug design utilizes apo-protein or complex structures retrieved from the PDB. >57% of crystallographic PDB entries were obtained with polyethyleneglycols (PEGs) as precipitant and/or as cryoprotectant, but >6% of these report presence of individual ethyleneglycol oligomers. We report a case in which ethyleneglycol oligomers' presence in a crystal structure markedly affected the bound ligand's position. Specifically, we compared the positions of methylene blue and decamethonium in acetylcholinesterase complexes obtained using isomorphous crystals precipitated with PEG200 or ammonium sulfate. The ligands' positions within the active-site gorge in complexes obtained using PEG200 are influenced by presence of ethyleneglycol oligomers in both cases bound to W84 at the gorge's bottom, preventing interaction of the ligand's proximal quaternary group with its indole. Consequently, both ligands are approximately 3.0A further up the gorge than in complexes obtained using crystals precipitated with ammonium sulfate, in which the quaternary groups make direct pi-cation interactions with the indole. These findings have implications for structure-based drug design, since data for ligand-protein complexes with polyethyleneglycol as precipitant may not reflect the ligand's position in its absence, and could result in selecting incorrect drug discovery leads. Docking methylene blue into the structure obtained with PEG200, but omitting the ethyleneglycols, yields results agreeing poorly with the crystal structure; excellent agreement is obtained if they are included. Many proteins display features in which precipitants might lodge. It will be important to investigate presence of precipitants in published crystal structures, and whether it has resulted in misinterpreting electron density maps, adversely affecting drug design. This article is protected by copyright. All rights reserved.
ESTHER : Dym_2016_Protein.Sci_25_1096
PubMedSearch : Dym_2016_Protein.Sci_25_1096
PubMedID: 26990888
Gene_locus related to this paper: torca-ACHE

Title : Automated Structure- and Sequence-Based Design of Proteins for High Bacterial Expression and Stability - Goldenzweig_2016_Mol.Cell_63_337
Author(s) : Goldenzweig A , Goldsmith M , Hill SE , Gertman O , Laurino P , Ashani Y , Dym O , Unger T , Albeck S , Prilusky J , Lieberman RL , Aharoni A , Silman I , Sussman JL , Tawfik DS , Fleishman SJ
Ref : Mol Cell , 63 :337 , 2016
Abstract : Upon heterologous overexpression, many proteins misfold or aggregate, thus resulting in low functional yields. Human acetylcholinesterase (hAChE), an enzyme mediating synaptic transmission, is a typical case of a human protein that necessitates mammalian systems to obtain functional expression. We developed a computational strategy and designed an AChE variant bearing 51 mutations that improved core packing, surface polarity, and backbone rigidity. This variant expressed at approximately 2,000-fold higher levels in E. coli compared to wild-type hAChE and exhibited 20 degrees C higher thermostability with no change in enzymatic properties or in the active-site configuration as determined by crystallography. To demonstrate broad utility, we similarly designed four other human and bacterial proteins. Testing at most three designs per protein, we obtained enhanced stability and/or higher yields of soluble and active protein in E. coli. Our algorithm requires only a 3D structure and several dozen sequences of naturally occurring homologs, and is available at http://pross.weizmann.ac.il.
ESTHER : Goldenzweig_2016_Mol.Cell_63_337
PubMedSearch : Goldenzweig_2016_Mol.Cell_63_337
PubMedID: 27425410
Gene_locus related to this paper: human-ACHE

Title : Catalytic metal ion rearrangements underline promiscuity and evolvability of a metalloenzyme - Ben-David_2013_J.Mol.Biol_425_1028
Author(s) : Ben-David M , Wieczorek G , Elias M , Silman I , Sussman JL , Tawfik DS
Ref : Journal of Molecular Biology , 425 :1028 , 2013
Abstract : Although largely deemed as structurally conserved, catalytic metal ion sites can rearrange, thereby contributing to enzyme evolvability. Here, we show that in paraoxonase-1, a lipo-lactonase, catalytic promiscuity and divergence into an organophosphate hydrolase are correlated with an alternative mode of the catalytic Ca(2+). We describe the crystal structures of active-site mutants bearing mutations at position 115. The histidine at this position acts as a base to activate the lactone-hydrolyzing water molecule. Mutations to Trp or Gln indeed diminish paraoxonase-1's lactonase activity; however, the promiscuous organophosphate hydrolase activity is enhanced. The structures reveal a 1.8-A upward displacement towards the enzyme's surface of the catalytic Ca(2+) in the His115 mutants and configurational changes in the ligating side chains and water molecules, relative to the wild-type enzyme. Biochemical analysis and molecular dynamics simulations suggest that this alternative, upward metal mode mediates the promiscuous hydrolysis of organophosphates. The upward Ca(2+) mode observed in the His115 mutants also appears to mediate the wild type's paraoxonase activity. However, whereas the upward mode dominates in the Trp115 mutant, it is scarcely populated in wild type. Thus, the plasticity of active-site metal ions may permit alternative, latent, promiscuous activities and also provide the basis for the divergence of new enzymatic functions.
ESTHER : Ben-David_2013_J.Mol.Biol_425_1028
PubMedSearch : Ben-David_2013_J.Mol.Biol_425_1028
PubMedID: 23318950

Title : The specific interaction of the photosensitizer methylene blue with acetylcholinesterase provides a model system for studying the molecular consequences of photodynamic therapy - Silman_2013_Chem.Biol.Interact_203_63
Author(s) : Silman I , Roth E , Paz A , Triquigneaux MM , Ehrenshaft M , Xu Y , Shnyrov VL , Sussman JL , Deterding LJ , Ashani Y , Mason RP , Weiner L
Ref : Chemico-Biological Interactions , 203 :63 , 2013
Abstract : The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a approximately 8 degrees C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.
ESTHER : Silman_2013_Chem.Biol.Interact_203_63
PubMedSearch : Silman_2013_Chem.Biol.Interact_203_63
PubMedID: 23159732

Title : Catalytic versatility and backups in enzyme active sites: the case of serum paraoxonase 1 - Ben-David_2012_J.Mol.Biol_418_181
Author(s) : Ben-David M , Elias M , Filippi JJ , Dunach E , Silman I , Sussman JL , Tawfik DS
Ref : Journal of Molecular Biology , 418 :181 , 2012
Abstract : The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1 (PON1) whose native substrates are lipophilic lactones. We describe the crystal structures of PON1 at a catalytically relevant pH and of its complex with a lactone analogue. The various PON1 structures and the analysis of active-site mutants guided the generation of docking models of the various substrates and their reaction intermediates. The models suggest that promiscuity is driven by coincidental overlaps between the reactive intermediate for the native lactonase reaction and the ground and/or intermediate states of the promiscuous reactions. This overlap is also enabled by different active-site conformations: the lactonase activity utilizes one active-site conformation whereas the promiscuous phosphotriesterase activity utilizes another. The hydrolysis of phosphotriesters, and of the aromatic lactone dihydrocoumarin, is also driven by an alternative catalytic mode that uses only a subset of the active-site residues utilized for lactone hydrolysis. Indeed, PON1's active site shows a remarkable level of networking and versatility whereby multiple residues share the same task and individual active-site residues perform multiple tasks (e.g., binding the catalytic calcium and activating the hydrolytic water). Overall, the coexistence of multiple conformations and alternative catalytic modes within the same active site underlines PON1's promiscuity and evolutionary potential.
ESTHER : Ben-David_2012_J.Mol.Biol_418_181
PubMedSearch : Ben-David_2012_J.Mol.Biol_418_181
PubMedID: 22387469

Title : Structural and functional characterization of the interaction of the photosensitizing probe methylene blue with Torpedo californica acetylcholinesterase - Paz_2012_Protein.Sci_21_1138
Author(s) : Paz A , Roth E , Ashani Y , Xu Y , Shnyrov VL , Sussman JL , Silman I , Weiner L
Ref : Protein Science , 21 :1138 , 2012
Abstract : The photosensitizer methylene blue MB generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase TcAChE In the dark it inhibits reversibly Binding is accompanied by a bathochromic absorption shift used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic anionic subsite CAS the peripheral anionic subsite PAS or bridging them MB is a noncompetitive inhibitor of TcAChE competing with reversible inhibitors directed at both anionic subsites but a single site is involved in inhibition MB also quenches TcAChE's intrinsic fluorescence It binds to TcAChE covalently inhibited by a small organophosphate OP but not an OP containing a bulky pyrene Differential scanning calorimetry shows an 8 degrees increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE and a less than twofold increase in cooperativity of the transition The crystal structure reveals a single MB stacked against Trp279 in the PAS oriented down the gorge toward the CAS it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330 midway down the gorge in the MB/TcAChE crystal structure precludes simultaneous binding of a second MB at the CAS Conversely binding of ligands at the CAS dislodges MB from its preferred locus at the PAS The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.
ESTHER : Paz_2012_Protein.Sci_21_1138
PubMedSearch : Paz_2012_Protein.Sci_21_1138
PubMedID: 22674800
Gene_locus related to this paper: torca-ACHE

Title : Backbone and side chain NMR assignments for the intrinsically disordered cytoplasmic domain of human neuroligin-3 - Wood_2012_Biomol.NMR.Assign_6_15
Author(s) : Wood K , Paz A , Dijkstra K , Scheek RM , Otten R , Silman I , Sussman JL , Mulder FA
Ref : Biomol NMR Assign , 6 :15 , 2012
Abstract : Neuroligins act as heterophilic adhesion molecules at neuronal synapses. Their cytoplasmic domains interact with synaptic scaffolding proteins, and have been shown to be intrinsically disordered. Here we report the backbone and side chain (1)H, (13)C and (15)N resonance assignments for the cytoplasmic domain of human neuroligin 3.
ESTHER : Wood_2012_Biomol.NMR.Assign_6_15
PubMedSearch : Wood_2012_Biomol.NMR.Assign_6_15
PubMedID: 21647611

Title : Evolved stereoselective hydrolases for broad-spectrum G-type nerve agent detoxification - Goldsmith_2012_Chem.Biol_19_456
Author(s) : Goldsmith M , Ashani Y , Simo Y , Ben-David M , Leader H , Silman I , Sussman JL , Tawfik DS
Ref : Chemical Biology , 19 :456 , 2012
Abstract : A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S(P) isomers of the most threatening G-type nerve agents. The evolved variants show <=340-fold increased rates and catalytic efficiencies of 0.2-5 x 10(7) M(-1) min(-1). Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) < 6.3 x 10(-4) in favor of the R(P) isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the S(P) isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics.
ESTHER : Goldsmith_2012_Chem.Biol_19_456
PubMedSearch : Goldsmith_2012_Chem.Biol_19_456
PubMedID: 22520752

Title : Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen: identification of N-formylkynurenine tryptophan derivatives within the active-site gorge of its complex with the photosensitizer Methylene Blue - Triquigneaux_2012_Biochem.J_448_83
Author(s) : Triquigneaux MM , Ehrenshaft M , Roth E , Silman I , Ashani Y , Mason RP , Weiner L , Deterding LJ
Ref : Biochemical Journal , 448 :83 , 2012
Abstract : The principal role of AChE (acetylcholinesterase) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The active site of AChE is near the bottom of a long and narrow gorge lined with aromatic residues. It contains a CAS (catalytic 'anionic' subsite) and a second PAS (peripheral 'anionic' site), the gorge mouth, both of which bind acetylcholine via pi-cation interactions, primarily with two conserved tryptophan residues. It was shown previously that generation of 1O2 by illumination of MB (Methylene Blue) causes irreversible inactivation of TcAChE (Torpedo californica AChE), and suggested that photo-oxidation of tryptophan residues might be responsible. In the present study, structural modification of the TcAChE tryptophan residues induced by MB-sensitized oxidation was investigated using anti-N-formylkynurenine antibodies and MS. From these analyses, we determined that N-formylkynurenine derivatives were specifically produced from Trp84 and Trp279, present at the CAS and PAS respectively. Peptides containing these two oxidized tryptophan residues were not detected when the competitive inhibitors, edrophonium and propidium (which should displace MB from the gorge) were present during illumination, in agreement with their efficient protection against the MB-induced photo-inactivation. Thus the bound MB elicited selective action of 1O2 on the tryptophan residues facing on to the water-filled active-site gorge. The findings of the present study thus demonstrate the localized action and high specificity of MB-sensitized photo-oxidation of TcAChE, as well as the value of this enzyme as a model system for studying the mechanism of action and specificity of photosensitizing agents.
ESTHER : Triquigneaux_2012_Biochem.J_448_83
PubMedSearch : Triquigneaux_2012_Biochem.J_448_83
PubMedID: 22888904

Title : Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis - Khare_2012_Nat.Chem.Biol_8_294
Author(s) : Khare SD , Kipnis Y , Greisen P, Jr. , Takeuchi R , Ashani Y , Goldsmith M , Song Y , Gallaher JL , Silman I , Leader H , Sussman JL , Stoddard BL , Tawfik DS , Baker D
Ref : Nat Chemical Biology , 8 :294 , 2012
Abstract : The ability to redesign enzymes to catalyze noncognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of metalloenzyme active site functional groups to catalyze new reactions. Using this method, we engineered a zinc-containing mouse adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency (k(cat)/K(m)) of ~10(4) M(-1) s(-1) after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzes the hydrolysis of the R(P) isomer of a coumarinyl analog of the nerve agent cyclosarin, and it shows marked substrate selectivity for coumarinyl leaving groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.
ESTHER : Khare_2012_Nat.Chem.Biol_8_294
PubMedSearch : Khare_2012_Nat.Chem.Biol_8_294
PubMedID: 22306579

Title : Backdoor opening mechanism in acetylcholinesterase based on X-ray crystallography and molecular dynamics simulations - Sanson_2011_Protein.Sci_20_1114
Author(s) : Sanson B , Colletier JP , Xu Y , Lang PT , Jiang H , Silman I , Sussman JL , Weik M
Ref : Protein Science , 20 :1114 , 2011
Abstract : The transient opening of a backdoor in the active-site wall of acetylcholinesterase, one of nature's most rapid enzymes, has been suggested to contribute to the efficient traffic of substrates and products. A crystal structure of Torpedo californica acetylcholinesterase in complex with the peripheral-site inhibitor aflatoxin is now presented, in which a tyrosine at the bottom of the active-site gorge rotates to create a 3.4-A wide exit channel. Molecular dynamics simulations show that the opening can be further enlarged by movement of Trp84. The crystallographic and molecular dynamics simulation data thus point to the interface between Tyr442 and Trp84 as the key element of a backdoor, whose opening permits rapid clearance of catalysis products from the active site. Furthermore, the crystal structure presented provides a novel template for rational design of inhibitors and reactivators, including anti-Alzheimer drugs and antidotes against organophosphate poisoning.
ESTHER : Sanson_2011_Protein.Sci_20_1114
PubMedSearch : Sanson_2011_Protein.Sci_20_1114
PubMedID: 21594947
Gene_locus related to this paper: torca-ACHE

Title : Directed evolution of hydrolases for prevention of G-type nerve agent intoxication - Gupta_2011_Nat.Chem.Biol_7_120
Author(s) : Gupta RD , Goldsmith M , Ashani Y , Simo Y , Mullokandov G , Bar H , Ben-David M , Leader H , Margalit R , Silman I , Sussman JL , Tawfik DS
Ref : Nat Chemical Biology , 7 :120 , 2011
Abstract : Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by approximately 10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) approximately 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.
ESTHER : Gupta_2011_Nat.Chem.Biol_7_120
PubMedSearch : Gupta_2011_Nat.Chem.Biol_7_120
PubMedID: 21217689

Title : Targeted oxidation of Torpedo californica acetylcholinesterase by singlet oxygen - Weiner_2011_Photochem.Photobiol_87_308
Author(s) : Weiner L , Roth E , Silman I
Ref : Photochem Photobiol , 87 :308 , 2011
Abstract : The photosensitizer, methylene blue (MB), is a strong reversible inhibitor of Torpedo californica acetylcholinesterase (AChE) in the dark. Under illumination it causes irreversible inactivation. Loss of fluorescence of the singlet oxygen ((1)O(2)) trap, 9,10-dimethylanthracene, was retarded in the presence of AChE, and the rate of photo-inactivation was increased in the presence of D(2)O, indicating that inactivation was due to (1)O(2) generated by the photosensitizer. CD revealed slightly reduced far-UV ellipticity, and slightly enhanced binding of an amphiphilic probe, indicating limited unfolding of the photo-oxidized AChE. However, both near-UV ellipticity and intrinsic fluorescence were markedly reduced, suggesting photo-oxidative damage to tryptophans, (Trp) supported by appearance of novel emission peaks ascribed to N'-formylkynurenine and/or kynurenine. Like other partially unfolded forms, the photo-oxidized AChE was sensitive to proteolysis. Photosensitized inactivation produced exclusively chemically cross-linked dimers, whereas irradiation of a partially unfolded state generated higher-order oligomers. The active-site gorge of AChE contains Trp in inhibitor-binding sites that might be targets for photo-oxidation. Indeed, reversible inhibitors retard photo-inactivation, and photo-inactivation destroys their binding sites. An excess of AChE protects paraoxonase from photo-inactivation by sequestering the photosensitizer. Affinity photo-oxidation of AChE by MB thus provides a valuable model for studying site-specific photo-inactivation of enzymes in both fundamental and clinical contexts.
ESTHER : Weiner_2011_Photochem.Photobiol_87_308
PubMedSearch : Weiner_2011_Photochem.Photobiol_87_308
PubMedID: 21155827

Title : In vitro detoxification of cyclosarin in human blood pre-incubated ex vivo with recombinant serum paraoxonases - Ashani_2011_Toxicol.Lett_206_24
Author(s) : Ashani Y , Goldsmith M , Leader H , Silman I , Sussman JL , Tawfik DS
Ref : Toxicol Lett , 206 :24 , 2011
Abstract : An ex vivo protocol was developed to assay the antidotal capacity of rePON1 variants to protect endogenous acetylcholinesterase and butyrylcholinesterase in human whole blood against OP nerve agents. This protocol permitted us to address the relationship between blood rePON1 concentrations, their kinetic parameters, and the level of protection conferred by rePON1 on the cholinesterases in human blood, following a challenge with cyclosarin (GF). The experimental data thus obtained were in good agreement with the predicted percent residual activities of blood cholinesterases calculated on the basis of the rate constants for inhibition of human acetylcholinesterase and butyrylcholinesterase by GF, the concentration of the particular rePON1 variant, and its k(cat)/K(m) value for GF. This protocol thus provides a rapid and reliable ex vivo screening tool for identification of rePON1 bioscavenger candidates suitable for protection of humans against organophosphorus-based toxicants. The results also permitted the refinement of a mathematical model for estimating the efficacious dose of rePON1s variants required for prophylaxis in humans.
ESTHER : Ashani_2011_Toxicol.Lett_206_24
PubMedSearch : Ashani_2011_Toxicol.Lett_206_24
PubMedID: 21807078

Title : Stereo-specific synthesis of analogs of nerve agents and their utilization for selection and characterization of paraoxonase (PON1) catalytic scavengers - Ashani_2010_Chem.Biol.Interact_187_362
Author(s) : Ashani Y , Gupta RD , Goldsmith M , Silman I , Sussman JL , Tawfik DS , Leader H
Ref : Chemico-Biological Interactions , 187 :362 , 2010
Abstract : Fluorogenic organophosphate inhibitors of acetylcholinesterase (AChE) homologous in structure to nerve agents provide useful probes for high throughput screening of mammalian paraoxonase (PON1) libraries generated by directed evolution of an engineered PON1 variant with wild-type like specificity (rePON1). Wt PON1 and rePON1 hydrolyze preferentially the less-toxic R(P) enantiomers of nerve agents and of their fluorogenic surrogates containing the fluorescent leaving group, 3-cyano-7-hydroxy-4-methylcoumarin (CHMC). To increase the sensitivity and reliability of the screening protocol so as to directly select rePON1 clones displaying stereo-preference towards the toxic S(P) enantiomer, and to determine accurately K(m) and k(cat) values for the individual isomers, two approaches were used to obtain the corresponding S(P) and R(P) isomers: (a) stereo-specific synthesis of the O-ethyl, O-n-propyl, and O-i-propyl analogs and (b) enzymic resolution of a racemic mixture of O-cyclohexyl methylphosphonylated CHMC. The configurational assignments of the S(P) and R(P) isomers, as well as their optical purity, were established by X-ray diffraction, reaction with sodium fluoride, hydrolysis by selected rePON1 variants, and inhibition of AChE. The S(P) configuration of the tested surrogates was established for the enantiomer with the more potent anti-AChE activity, with S(P)/R(P) inhibition ratios of 10-100, whereas the R(P) isomers of the O-ethyl and O-n-propyl were hydrolyzed by wt rePON1 about 600- and 70-fold faster, respectively, than the S(P) counterpart. Wt rePON1-induced R(P)/S(P) hydrolysis ratios for the O-cyclohexyl and O-i-propyl analogs are estimated to be >>1000. The various S(P) enantiomers of O-alkyl-methylphosphonyl esters of CHMC provide suitable ligands for screening rePON1 libraries, and can expedite identification of variants with enhanced catalytic proficiency towards the toxic nerve agents.
ESTHER : Ashani_2010_Chem.Biol.Interact_187_362
PubMedSearch : Ashani_2010_Chem.Biol.Interact_187_362
PubMedID: 20303930

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 : Long route or shortcut? A molecular dynamics study of traffic of thiocholine within the active-site gorge of acetylcholinesterase - Xu_2010_Biophys.J_99_4003
Author(s) : Xu Y , Colletier JP , Weik M , Qin G , Jiang H , Silman I , Sussman JL
Ref : Biophysical Journal , 99 :4003 , 2010
Abstract : The principal role of acetylcholinesterase is termination of nerve impulse transmission at cholinergic synapses, by rapid hydrolysis of the neurotransmitter acetylcholine to acetate and choline. Its active site is buried at the bottom of a deep and narrow gorge, at the rim of which is found a second anionic site, the peripheral anionic site. The fact that the active site is so deeply buried has raised cogent questions as to how rapid traffic of substrate and products occurs in such a confined environment. Various theoretical and experimental approaches have been used to solve this problem. Here, multiple conventional molecular dynamics simulations have been performed to investigate the clearance of the product, thiocholine, from the active-site gorge of acetylcholinesterase. Our results indicate that thiocholine is released from the peripheral anionic site via random pathways, while three exit routes appear to be favored for its release from the active site, namely, along the axis of the active-site gorge, and through putative back- and side-doors. The back-door pathway is that via which thiocholine exits most frequently. Our results are in good agreement with kinetic and kinetic-crystallography studies. We propose the use of multiple molecular dynamics simulations as a fast yet accurate complementary tool in structural studies of enzymatic trafficking.
ESTHER : Xu_2010_Biophys.J_99_4003
PubMedSearch : Xu_2010_Biophys.J_99_4003
PubMedID: 21156143

Title : Amalgam, an axon guidance Drosophila adhesion protein belonging to the immunoglobulin superfamily: over-expression, purification and biophysical characterization - Zeev-Ben-Mordehai_2009_Protein.Expr.Purif_63_147
Author(s) : Zeev-Ben-Mordehai T , Paz A , Peleg Y , Toker L , Wolf SG , Rydberg EH , Sussman JL , Silman I
Ref : Protein Expr Purif , 63 :147 , 2009
Abstract : Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote adhesion. Amalgam was heterologously expressed in Pichia pastoris, and the secreted protein product, bearing an NH(2)-terminal His(6)Tag, was purified from the growth medium by metal affinity chromatography. Size exclusion chromatography separated the purified protein into two fractions: a major, multimeric fraction and a minor, dimeric one. Two protocols to reduce the percentage of multimers were tested. In one, protein induction was performed in the presence of the zwitterionic detergent CHAPS, yielding primarily the dimeric form of amalgam. In a second protocol, agitation was gradually reduced during the course of the induction and antifoam was added daily to reduce the air/liquid interfacial foam area. This latter protocol lowered the percentage of multimer 2-fold, compared to constant agitation. Circular dichroism measurements showed that the dimeric fraction had a high beta-sheet content, as expected for a protein with an immunoglobulin fold. Dynamic light scattering and sedimentation velocity measurements showed that the multimeric fraction displays a monodisperse distribution, with R(H)=16 nm. When co-expressed together with amalgam the ectodomain of neurotactin copurified with it. Furthermore, both purified fractions of amalgam were shown to interact with Torpedo californica acetylcholinesterase, a structural homolog of neurotactin.
ESTHER : Zeev-Ben-Mordehai_2009_Protein.Expr.Purif_63_147
PubMedSearch : Zeev-Ben-Mordehai_2009_Protein.Expr.Purif_63_147
PubMedID: 18938249

Title : Directed evolution of serum paraoxonase PON3 by family shuffling and ancestor\/consensus mutagenesis, and its biochemical characterization - Khersonsky_2009_Biochemistry_48_6644
Author(s) : Khersonsky O , Rosenblat M , Toker L , Yacobson S , Hugenmatter A , Silman I , Sussman JL , Aviram M , Tawfik DS
Ref : Biochemistry , 48 :6644 , 2009
Abstract : Serum paraoxonases (PONs) are calcium-dependent lactonases with anti-atherogenic and detoxification functions. Here we describe the directed evolution and characterization of recombinant variants of serum paraoxonase PON3 that express in an active and soluble manner in Escherichia coli. These variants were obtained by combining family shuffling and phylogeny-based mutagenesis: the limited diversity of accessible, cloned PON3 genes was complemented by spiking the shuffling reaction with ancestor/consensus mutations, mutations to residues that comprise the consensus or appear in the predicted ancestors of the PON family. We screened the resulting libraries for PON3's lactonase activity while ensuring that the selected variants retained the substrate specificity of wild-type mammalian PON3s. The availability of highly stable, recombinant PON3 that is free of all other serum components enabled us to explore unknown biochemical features of PON3, including its binding to HDL particles, the effect of HDL on PON3's stability and enzymatic activity, and ex vivo tests of its anti-atherogenic properties. Overall, it appears that PON3 possesses properties very similar to those of PON1: the enzyme's lactonase activity is selectively stimulated by binding to apoAI-HDL, with a concomitant increase in its stability. PON3 also exhibits potentially anti-atherogenic functions, although at levels lower than those of PON1.
ESTHER : Khersonsky_2009_Biochemistry_48_6644
PubMedSearch : Khersonsky_2009_Biochemistry_48_6644
PubMedID: 19492856

Title : The crystal structure of a complex of acetylcholinesterase with a bis-(-)-nor-meptazinol derivative reveals disruption of the catalytic triad - Paz_2009_J.Med.Chem_52_2543
Author(s) : Paz A , Xie Q , Greenblatt HM , Fu W , Tang Y , Silman I , Qiu Z , Sussman JL
Ref : Journal of Medicinal Chemistry , 52 :2543 , 2009
Abstract : A bis-(-)-nor-meptazinol derivative in which the two meptazinol rings are linked by a nonamethylene spacer is a novel acetylcholinesterase inhibitor that inhibits both catalytic activity and Abeta peptide aggregation. The crystal structure of its complex with Torpedo californica acetylcholinesterase was determined to 2.7 A resolution. The ligand spans the active-site gorge, with one nor-meptazinol moiety bound at the "anionic" subsite of the active site, disrupting the catalytic triad by forming a hydrogen bond with His440N(epsilon2), which is hydrogen-bonded to Ser200O(gamma) in the native enzyme. The second nor-meptazinol binds at the peripheral "anionic" site at the gorge entrance. A number of GOLD models of the complex, using both native TcAChE and the protein template from the crystal structure of the bis-(-)-nor-meptazinol/TcAChE complex, bear higher similarity to the X-ray structure than a previous model obtained using the mouse enzyme structure. These findings may facilitate rational design of new meptazinol-based acetylcholinesterase inhibitors.
ESTHER : Paz_2009_J.Med.Chem_52_2543
PubMedSearch : Paz_2009_J.Med.Chem_52_2543
PubMedID: 19326912
Gene_locus related to this paper: torca-ACHE

Title : The quaternary structure of amalgam, a Drosophila neuronal adhesion protein, explains its dual adhesion properties - Zeev-Ben-Mordehai_2009_Biophys.J_97_2316
Author(s) : Zeev-Ben-Mordehai T , Mylonas E , Paz A , Peleg Y , Toker L , Silman I , Svergun DI , Sussman JL
Ref : Biophysical Journal , 97 :2316 , 2009
Abstract : Amalgam (Ama) is a secreted neuronal adhesion protein that contains three tandem immunoglobulin domains. It has both homophilic and heterophilic cell adhesion properties, and is required for axon guidance and fasciculation during early stages of Drosophila development. Here, we report its biophysical characterization and use small-angle x-ray scattering to determine its low-resolution structure in solution. The biophysical studies revealed that Ama forms dimers in solution, and that its secondary and tertiary structures are typical for the immunoglobulin superfamily. Ab initio and rigid-body modeling by small-angle x-ray scattering revealed a distinct V-shaped dimer in which the two monomer chains are aligned parallel to each other, with the dimerization interface being formed by domain 1. These data provide a structural basis for the dual adhesion characteristics of Ama. Thus, the dimeric structure explains its homophilic adhesion properties. Its V shape suggests a mechanism for its interaction with its receptor, the single-pass transmembrane adhesion protein neurotactin, in which each "arm" of Ama binds to the extracellular domain of neurotactin, thus promoting its clustering on the outer face of the plasma membrane.
ESTHER : Zeev-Ben-Mordehai_2009_Biophys.J_97_2316
PubMedSearch : Zeev-Ben-Mordehai_2009_Biophys.J_97_2316
PubMedID: 19843464

Title : Stabilization of Torpedo californica acetylcholinesterase by reversible inhibitors - Weiner_2009_Biochemistry_48_563
Author(s) : Weiner L , Shnyrov VL , Konstantinovskii L , Roth E , Ashani Y , Silman I
Ref : Biochemistry , 48 :563 , 2009
Abstract : The dimeric form of Torpedo californica acetylcholinesterase provides a valuable experimental system for studying transitions between native, partially unfolded, and unfolded states since long-lived partially unfolded states can be generated by chemical modification of a nonconserved buried cysteine residue, Cys 231, by denaturing agents, by oxidative stress, and by thermal inactivation. Elucidation of the 3D structures of complexes of Torpedo californica acetylcholinesterase with a repertoire of reversible inhibitors permits their classification into three categories: (a) active-site directed inhibitors, which interact with the catalytic anionic subsite, at the bottom of the active-site gorge, such as edrophonium and tacrine; (b) peripheral anionic site inhibitors, which interact with a site at the entrance to the gorge, such as propidium and d-tubocurarine; and (c) elongated gorge-spanning inhibitors, which bridge the two sites, such as BW284c51 and decamethonium. The effects of these three categories of reversible inhibitors on the stability of Torpedo californica acetylcholinesterase were investigated using spectroscopic techniques and differential scanning calorimetry. Thermodynamic parameters obtained calorimetrically permitted quantitative comparison of the effects of the inhibitors on the enzyme's thermal stability. Peripheral site inhibitors had a relatively small effect, while gorge-spanning ligands and those binding at the catalytic anionic site, had a much larger stabilizing effect. The strongest effect was, however, observed with the polypeptide toxin, fasciculin II (FasII), even though, in terms of its binding site, it belongs to the category of peripheral site ligands. The stabilizing effect of the ligands binding at the anionic subsite of the active site, like that of the gorge-spanning ligands, may be ascribed to their capacity to stabilize the interaction between the two subdomains of the enzyme. The effect of fasciculin II may be ascribed to the large surface area of interaction (>2000 A(2)) between the two proteins. Stabilization of Torpedo californica acetylcholinesterase by both divalent cations and chemical chaperones was earlier shown to be due to a shift in equilibrium between the native state and a partially unfolded state ( Millard et al. ( 2003 ) Protein Sci. 12 , 2337 - 2347 ). The low molecular weight inhibitors used in the present study may act similarly and can thus be considered as pharmacological chaperones for stabilizing the fully folded native form of the enzyme.
ESTHER : Weiner_2009_Biochemistry_48_563
PubMedSearch : Weiner_2009_Biochemistry_48_563
PubMedID: 19115961

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

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

Title : 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 : Shoot-and-Trap: use of specific x-ray damage to study structural protein dynamics by temperature-controlled cryo-crystallography - Colletier_2008_Proc.Natl.Acad.Sci.U.S.A_105_11742
Author(s) : Colletier JP , Bourgeois D , Sanson B , Fournier D , Sussman JL , Silman I , Weik M
Ref : Proc Natl Acad Sci U S A , 105 :11742 , 2008
Abstract : Although x-ray crystallography is the most widely used method for macromolecular structure determination, it does not provide dynamical information, and either experimental tricks or complementary experiments must be used to overcome the inherently static nature of crystallographic structures. Here we used specific x-ray damage during temperature-controlled crystallographic experiments at a third-generation synchrotron source to trigger and monitor (Shoot-and-Trap) structural changes putatively involved in an enzymatic reaction. In particular, a nonhydrolyzable substrate analogue of acetylcholinesterase, the "off-switch" at cholinergic synapses, was radiocleaved within the buried enzymatic active site. Subsequent product clearance, observed at 150 K but not at 100 K, indicated exit from the active site possibly via a "backdoor." The simple strategy described here is, in principle, applicable to any enzyme whose structure in complex with a substrate analogue is available and, therefore, could serve as a standard procedure in kinetic crystallography studies.
ESTHER : Colletier_2008_Proc.Natl.Acad.Sci.U.S.A_105_11742
PubMedSearch : Colletier_2008_Proc.Natl.Acad.Sci.U.S.A_105_11742
PubMedID: 18701720
Gene_locus related to this paper: torca-ACHE

Title : Acetylcholinesterase: how is structure related to function? - Silman_2008_Chem.Biol.Interact_175_3
Author(s) : Silman I , Sussman JL
Ref : Chemico-Biological Interactions , 175 :3 , 2008
Abstract : In accordance with its biological role, termination of neurotransmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine, acetylcholinesterase is one of nature's most efficient enzymes. Solution of its three-dimensional structure revealed that its active site is located at the bottom of a deep and narrow gorge. Such an architecture was unanticipated in view of its high turnover number. The present review examines how the highly specialized structure of acetylcholinesterase, with its sequestered active site, contributes to its catalytic efficacy, and discusses how the traffic of substrate and products to and from the active site is controlled.
ESTHER : Silman_2008_Chem.Biol.Interact_175_3
PubMedSearch : Silman_2008_Chem.Biol.Interact_175_3
PubMedID: 18586019

Title : Biophysical characterization of the unstructured cytoplasmic domain of the human neuronal adhesion protein neuroligin 3 - Paz_2008_Biophys.J_95_1928
Author(s) : Paz A , Zeev-Ben-Mordehai T , Lundqvist M , Sherman E , Mylonas E , Weiner L , Haran G , Svergun DI , Mulder FA , Sussman JL , Silman I
Ref : Biophysical Journal , 95 :1928 , 2008
Abstract : Cholinesterase-like adhesion molecules (CLAMs) are a family of neuronal cell adhesion molecules with important roles in synaptogenesis, and in maintaining structural and functional integrity of the nervous system. Our earlier study on the cytoplasmic domain of one of these CLAMs, the Drosophila protein, gliotactin, showed that it is intrinsically unstructured in vitro. Bioinformatic analysis suggested that the cytoplasmic domains of other CLAMs are also intrinsically unstructured, even though they bear no sequence homology to each other or to any known protein. In this study, we overexpress and purify the cytoplasmic domain of human neuroligin 3, notwithstanding its high sensitivity to the Escherichia coli endogenous proteases that cause its rapid degradation. Using bioinformatic analysis, sensitivity to proteases, size exclusion chromatography, fluorescence correlation spectroscopy, analytical ultracentrifugation, small angle x-ray scattering, circular dichroism, electron spin resonance, and nuclear magnetic resonance, we show that the cytoplasmic domain of human neuroligin 3 is intrinsically unstructured. However, several of these techniques indicate that it is not fully extended, but becomes significantly more extended under denaturing conditions.
ESTHER : Paz_2008_Biophys.J_95_1928
PubMedSearch : Paz_2008_Biophys.J_95_1928
PubMedID: 18456828

Title : Induced-fit or preexisting equilibrium dynamics? Lessons from protein crystallography and MD simulations on acetylcholinesterase and implications for structure-based drug design - Xu_2008_Protein.Sci_17_601
Author(s) : Xu Y , Colletier JP , Jiang H , Silman I , Sussman JL , Weik M
Ref : Protein Science , 17 :601 , 2008
Abstract : Crystal structures of acetylcholinesterase complexed with ligands are compared with side-chain conformations accessed by native acetylcholinesterase in molecular dynamics (MD) simulations. Several crystallographic conformations of a key residue in a specific binding site are accessed in a simulation of native acetylcholinesterase, although not seen in rotomer plots. Conformational changes upon ligand binding thus involve preexisting equilibrium dynamics. Consequently, rational drug design could benefit significantly from conformations monitored by MD simulations of native targets.
ESTHER : Xu_2008_Protein.Sci_17_601
PubMedSearch : Xu_2008_Protein.Sci_17_601
PubMedID: 18359854

Title : Flexibility of aromatic residues in the active-site gorge of acetylcholinesterase: X-ray versus molecular dynamics - Xu_2008_Biophys.J_95_2500
Author(s) : Xu Y , Colletier JP , Weik M , Jiang H , Moult J , Silman I , Sussman JL
Ref : Biophysical Journal , 95 :2500 , 2008
Abstract : The high aromatic content of the deep and narrow active-site gorge of acetylcholinesterase (AChE) is a remarkable feature of this enzyme. Here, we analyze conformational flexibility of the side chains of the 14 conserved aromatic residues in the active-site gorge of Torpedo californica AChE based on the 47 three-dimensional crystal structures available for the native enzyme, and for its complexes and conjugates, and on a 20-ns molecular dynamics (MD) trajectory of the native enzyme. The degree of flexibility of these 14 aromatic side chains is diverse. Although the side-chain conformations of F330 and W279 are both very flexible, the side-chain conformations of F120, W233, W432, Y70, Y121, F288, F290 and F331 appear to be fixed. Residues located on, or adjacent to, the Omega-loop (C67-C94), namely W84, Y130, Y442, and Y334, display different flexibilities in the MD simulations and in the crystal structures. An important outcome of our study is that the majority of the side-chain conformations observed in the 47 Torpedo californica AChE crystal structures are faithfully reproduced by the MD simulation on the native enzyme. Thus, the protein can assume these conformations even in the absence of the ligand that permitted their experimental detection. These observations are pertinent to structure-based drug design.
ESTHER : Xu_2008_Biophys.J_95_2500
PubMedSearch : Xu_2008_Biophys.J_95_2500
PubMedID: 18502801

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

Title : Bivalent ligands derived from Huperzine A as acetylcholinesterase inhibitors - Haviv_2007_Curr.Top.Med.Chem_7_375
Author(s) : Haviv H , Wong DM , Silman I , Sussman JL
Ref : Curr Top Med Chem , 7 :375 , 2007
Abstract : The naturally occurring alkaloid Huperzine A (HupA) is an acetylcholinesterase (AChE) inhibitor that has been used for centuries as a Chinese folk medicine in the context of its source plant Huperzia Serrata. The potency and relative safety of HupA rendered it a promising drug for the ameliorative treatment of Alzheimer's disease (AD) vis-a-vis the "cholinergic hypothesis" that attributes the cognitive decrements associated with AD to acetylcholine deficiency in the brain. However, recent evidence supports a neuroprotective role for HupA, suggesting that it could act as more than a mere palliative. Biochemical and crystallographic studies of AChE revealed two potential binding sites in the active-site gorge of AChE, one of which, the "peripheral anionic site" at the mouth of the gorge, was implicated in promoting aggregation of the beta amyloid (Abeta) peptide responsible for the neurodegenerative process in AD. This feature of AChE facilitated the development of dual-site binding HupA-based bivalent ligands, in hopes of concomitantly increasing AChE inhibition potency by utilizing the "chelate effect", and protecting neurons from Abeta toxicity. Crystal structures of AChE allowed detailed modeling and docking studies that were instrumental in enhancing the understanding of underlying principles of bivalent inhibitor-enzyme dynamics. This monograph reviews two categories of HupA-based bivalent ligands, in which HupA and HupA fragments serve as building blocks, with a focus on the recently solved crystallographic structures of Torpedo californica AChE in complex with such bifunctional agents. The advantages and drawbacks of such structured-based drug design, as well as species differences, are highlighted and discussed.
ESTHER : Haviv_2007_Curr.Top.Med.Chem_7_375
PubMedSearch : Haviv_2007_Curr.Top.Med.Chem_7_375
PubMedID: 17305579

Title : Complexes of alkylene-linked tacrine dimers with Torpedo californica acetylcholinesterase: Binding of Bis5-tacrine produces a dramatic rearrangement in the active-site gorge - Rydberg_2006_J.Med.Chem_49_5491
Author(s) : Rydberg EH , Brumshtein B , Greenblatt HM , Wong DM , Shaya D , Williams LD , Carlier PR , Pang YP , Silman I , Sussman JL
Ref : Journal of Medicinal Chemistry , 49 :5491 , 2006
Abstract : The X-ray crystal structures were solved for complexes with Torpedo californica acetylcholinesterase of two bivalent tacrine derivative compounds in which the two tacrine rings were separated by 5- and 7-carbon spacers. The derivative with the 7-carbon spacer spans the length of the active-site gorge, making sandwich interactions with aromatic residues both in the catalytic anionic site (Trp84 and Phe330) at the bottom of the gorge and at the peripheral anionic site near its mouth (Tyr70 and Trp279). The derivative with the 5-carbon spacer interacts in a similar manner at the bottom of the gorge, but the shorter tether precludes a sandwich interaction at the peripheral anionic site. Although the upper tacrine group does interact with Trp279, it displaces the phenyl residue of Phe331, thus causing a major rearrangement in the Trp279-Ser291 loop. The ability of this inhibitor to induce large-scale structural changes in the active-site gorge of acetylcholinesterase has significant implications for structure-based drug design because such conformational changes in the target enzyme are difficult to predict and to model.
ESTHER : Rydberg_2006_J.Med.Chem_49_5491
PubMedSearch : Rydberg_2006_J.Med.Chem_49_5491
PubMedID: 16942022
Gene_locus related to this paper: torca-ACHE

Title : Structural insights into substrate traffic and inhibition in acetylcholinesterase - Colletier_2006_EMBO.J_25_2746
Author(s) : Colletier JP , Fournier D , Greenblatt HM , Stojan J , Sussman JL , Zaccai G , Silman I , Weik M
Ref : EMBO Journal , 25 :2746 , 2006
Abstract : Acetylcholinesterase (AChE) terminates nerve-impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine. Substrate traffic in AChE involves at least two binding sites, the catalytic and peripheral anionic sites, which have been suggested to be allosterically related and involved in substrate inhibition. Here, we present the crystal structures of Torpedo californica AChE complexed with the substrate acetylthiocholine, the product thiocholine and a nonhydrolysable substrate analogue. These structures provide a series of static snapshots of the substrate en route to the active site and identify, for the first time, binding of substrate and product at both the peripheral and active sites. Furthermore, they provide structural insight into substrate inhibition in AChE at two different substrate concentrations. Our structural data indicate that substrate inhibition at moderate substrate concentration is due to choline exit being hindered by a substrate molecule bound at the peripheral site. At the higher concentration, substrate inhibition arises from prevention of exit of acetate due to binding of two substrate molecules within the active-site gorge.
ESTHER : Colletier_2006_EMBO.J_25_2746
PubMedSearch : Colletier_2006_EMBO.J_25_2746
PubMedID: 16763558
Gene_locus related to this paper: torca-ACHE

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

Title : The 3D structure of the anticancer prodrug CPT-11 with Torpedo californica acetylcholinesterase rationalizes its inhibitory action on AChE and its hydrolysis by butyrylcholinesterase and carboxylesterase - Harel_2005_Chem.Biol.Interact_157-158_153
Author(s) : Harel M , Hyatt JL , Brumshtein B , Morton CL , Wadkins RM , Silman I , Sussman JL , Potter PM
Ref : Chemico-Biological Interactions , 157-158 :153 , 2005
Abstract : The anticancer prodrug CPT-11 is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. The 2.6 angstroms resolution crystal structure of its complex with Torpedo californica acetylcholinesterase (TcAChE) demonstrates that CPT-11 binds to TcAChE and spans its gorge similarly to the Alzheimer drug, Aricept. The crystal structure clearly reveals the interactions, which contribute to the inhibitory action of CPT-11. Modeling of the complexes of CPT-11 with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug, shows how binding to either of the two enzymes yields a productive substrate-enzyme complex.
ESTHER : Harel_2005_Chem.Biol.Interact_157-158_153
PubMedSearch : Harel_2005_Chem.Biol.Interact_157-158_153
PubMedID: 16289500
Gene_locus related to this paper: torca-ACHE

Title : The crystal structure of the complex of the anticancer prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin (CPT-11) with Torpedo californica acetylcholinesterase provides a molecular explanation for its cholinergic action - Harel_2005_Mol.Pharmacol_67_1874
Author(s) : Harel M , Hyatt JL , Brumshtein B , Morton CL , Yoon KJ , Wadkins RM , Silman I , Sussman JL , Potter PM
Ref : Molecular Pharmacology , 67 :1874 , 2005
Abstract : The anticancer prodrug 7-ethyl-10-[4-(1-piperidino)-1-piperidino-]carbonyloxycamptothecin (CPT-11) is a highly effective camptothecin analog that has been approved for the treatment of colon cancer. It is hydrolyzed by carboxylesterases to yield 7-ethyl-10-hydroxycamptothecin (SN-38), a potent topoisomerase I poison. However, upon high-dose intravenous administration of CPT-11, a cholinergic syndrome is observed that can be ameliorated by atropine. Previous studies have indicated that CPT-11 can inhibit acetylcholinesterase (AChE), and here, we provide a detailed analysis of the inhibition of AChE by CPT-11 and by structural analogs. These studies demonstrate that the terminal dipiperidino moiety in CPT-11 plays a major role in enzyme inhibition, and this has been confirmed by X-ray crystallographic studies of a complex of the drug with Torpedo californica AChE. Our results indicate that CPT-11 binds within the active site gorge of the protein in a fashion similar to that observed with the Alzheimer drug donepezil. The 3D structure of the CPT-11/AChE complex also permits modeling of CPT-11 complexed with mammalian butyrylcholinesterase and carboxylesterase, both of which are known to hydrolyze the drug to the active metabolite. Overall, the results presented here clarify the mechanism of AChE inhibition by CPT-11 and detail the interaction of the drug with the protein. These studies may allow the design of both novel camptothecin analogs that would not inhibit AChE and new AChE inhibitors derived from the camptothecin scaffold.
ESTHER : Harel_2005_Mol.Pharmacol_67_1874
PubMedSearch : Harel_2005_Mol.Pharmacol_67_1874
PubMedID: 15772291
Gene_locus related to this paper: torca-ACHE

Title : Crystal packing mediates enantioselective ligand recognition at the peripheral site of acetylcholinesterase - Haviv_2005_J.Am.Chem.Soc_127_11029
Author(s) : Haviv H , Wong DM , Greenblatt HM , Carlier PR , Pang YP , Silman I , Sussman JL
Ref : Journal of the American Chemical Society , 127 :11029 , 2005
Abstract : Recently, alkylene-linked heterodimers of tacrine (1) and 5-amino-5,6,7,8-tetrahydroquinolinone (2, hupyridone) were shown to exhibit higher acetylcholinesterase (AChE) inhibition than either monomeric 1 or 2. Such inhibitors are potential drug candidates for ameliorating the cognitive decrements in early Alzheimer patients. In an attempt to understand the inhibition mechanism of one such dimer, (RS)-(+/-)-N-9-(1,2,3,4-tetrahydroacridinyl)-N'-5-[5,6,7,8-tetrahydro-2'(1'H)-qui nolinonyl]-1,10-diaminodecane [(RS)-(+/-)-3] bisoxalate, the racemate was soaked in trigonal Torpedo californica AChE (TcAChE) crystals, and the X-ray structure of the resulting complex was solved to 2.30 A resolution. Its structure revealed the 1 unit bound to the "anionic" subsite of the active site, near the bottom of the active-site gorge, as seen for the 1/TcAChE complex. Interestingly, only the (R)-enantiomer of the 2 unit was seen in the peripheral "anionic" site (PAS) at the top of the gorge, and was hydrogen-bonded to the side chains of residues belonging to an adjacent, symmetry-related AChE molecule covering the gorge entrance. When the same racemate was soaked in orthorhombic crystals of TcAChE, in which the entrance to the gorge is more exposed, the crystal structure of the corresponding complex revealed no substantial enantiomeric selectivity. This observation suggests that the apparent enantiomeric selectivity of trigonal crystals of TcAChE for (R)-3 is mainly due to crystal packing, resulting in preferential binding of one enantiomeric inhibitor both to its "host" enzyme and to its neighbor in the asymmetric unit, rather than to steric constraints imposed by the geometry of the active-site gorge.
ESTHER : Haviv_2005_J.Am.Chem.Soc_127_11029
PubMedSearch : Haviv_2005_J.Am.Chem.Soc_127_11029
PubMedID: 16076210
Gene_locus related to this paper: torca-ACHE

Title : Inhibition of acetylcholinesterase by the anticancer prodrug CPT-11 - Hyatt_2005_Chem.Biol.Interact_157-158_247
Author(s) : Hyatt JL , Tsurkan L , Morton CL , Yoon KJ , Harel M , Brumshtein B , Silman I , Sussman JL , Wadkins RM , Potter PM
Ref : Chemico-Biological Interactions , 157-158 :247 , 2005
Abstract : CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) is an anticancer prodrug that has been approved for the treatment of colon cancer. It is a member of the camptothecin class of drugs and activation to the active metabolite SN-38, is mediated by carboxylesterases (CE). SN-38 is a potent topoisomerase I poison and is highly effective at killing human tumor cells, with IC50 values in the low nM range. However, upon high dose administration of CPT-11 to cancer patients, a cholinergic syndrome is observed, that can be rapidly ameliorated by atropine. This suggests a direct interaction of the drug or its metabolites with acetylcholinesterase (AChE). Kinetic studies indicated that CPT-11 was primarily responsible for AChE inhibition with the 4-piperidinopiperidine moiety, the major determinant in the loss of enzyme activity. Structural analogs of 4-piperidinopiperidine however, did not inhibit AChE, including a benzyl piperazine derivate of CPT-11. These results suggest that novel anticancer drugs could be synthesized that do not inhibit AChE, or alternatively, that novel AChE inhibitors could be designed based around the camptothecin scaffold.
ESTHER : Hyatt_2005_Chem.Biol.Interact_157-158_247
PubMedSearch : Hyatt_2005_Chem.Biol.Interact_157-158_247
PubMedID: 16257398

Title : Analysis of genetic polymorphisms in acetylcholinesterase as reflected in different populations - Hasin_2005_Curr.Alzheimer.Res_2_207
Author(s) : Hasin Y , Avidan N , Bercovich D , Korczyn AD , Silman I , Beckmann JS , Sussman JL
Ref : Curr Alzheimer Res , 2 :207 , 2005
Abstract : Acetylcholinesterase (AChE) plays a crucial physiological role in termination of impulse transmission at cholinergic synapses through rapid hydrolysis of acetylcholine. In addition, it was implicated in amyloid plaque formation, a hallmark of Alzheimer's disease (AD), and most of the drugs used in AD treatment are AChE inhibitors. Thus ACHE is an obvious candidate gene for pharmacogenetic study of AD treatment. However, AChE is a highly conserved molecule, and only a few naturally occurring genetic polymorphisms have been reported in the human gene. The goals of this study were to make a systematic effort to identify natural single nucleotide polymorphisms (SNPs) in the human ACHE gene, and to reveal their population specific architecture. To this end, the genomic coding sequences for AChE of 96 unrelated control individuals from three distinct ethnic groups, African Americans, Ashkenazi Jews and Israeli Arabs, were analyzed. Thirteen ACHE SNPs were identified, ten of which are newly described, and five of which should produce amino-acid substitutions (Arg34Gln, Gly57Arg, Glu344Gly, His353Asn and Pro592Arg). Population frequencies of 11 of the 13 SNPs were established in four different populations, African Americans, Ashkenazi Jews, Sephardic Jews and Israeli Arabs; 17 haplotypes and 5 ethno-specific alleles were identified, and a cladogram of ACHE haplotypes was constructed. Among the SNPs resulting in an amino-acid substitution, three are within the mature protein, mapping on its external surface; they are thus unlikely to affect its catalytic properties, yet could have antigenic consequences or affect putative protein-protein interactions. Furthermore, the newly identified SNPs open the door to a study of the possible association of AChE with deleterious phenotypes - such as adverse drug responses to AChE inhibitors employed in treatment of AD patients and hypersensitivity to pesticides.
ESTHER : Hasin_2005_Curr.Alzheimer.Res_2_207
PubMedSearch : Hasin_2005_Curr.Alzheimer.Res_2_207
PubMedID: 15974920

Title : Dynamic mechanism of E2020 binding to acetylcholinesterase: a steered molecular dynamics simulation - Niu_2005_J.Phys.Chem.B_109_23730
Author(s) : Niu C , Xu Y , Luo X , Duan W , Silman I , Sussman JL , Zhu W , Chen K , Shen J , Jiang H
Ref : J Phys Chem B , 109 :23730 , 2005
Abstract : The unbinding process of E2020 ((R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methylpiperidine) leaving from the long active site gorge of Torpedo californica acetylcholinesterase (TcAChE) was studied by using steered molecular dynamics (SMD) simulations on a nanosecond scale with different velocities, and unbinding force profiles were obtained. Different from the unbinding of other AChE inhibitors, such as Huperzine A that undergoes the greatest barrier located at the bottleneck of the gorge, the major resistance preventing E2020 from leaving the gorge is from the peripheral anionic site where E2020 interacts intensively with several aromatic residues (e.g., Tyr70, Tyr121, and Trp279) through its benzene ring and forms a strong direct hydrogen bond and a water bridge with Ser286 via its O24. These interactions cause the largest rupture force, approximately 550 pN. It was found that the rotatable bonds of the piperidine ring to the benzene ring and dimethoxyindanone facilitate E2020 to pass the bottleneck through continuous conformation change by rotating those bonds to avoid serious conflict with Tyr121 and Phe330. The aromatic residues lining the gorge wall are the major components contributing to hydrophobic interactions between E2020 and TcAChE. Remarkably, these aromatic residues, acting in three groups as "sender" and "receiver", compose a "conveyer belt" for E2020 entering and leaving the TcAChE gorge.
ESTHER : Niu_2005_J.Phys.Chem.B_109_23730
PubMedSearch : Niu_2005_J.Phys.Chem.B_109_23730
PubMedID: 16375354

Title : Depolarization initiates phasic acetylcholine release by relief of a tonic block imposed by presynaptic M2 muscarinic receptors - Parnas_2005_J.Neurophysiol_93_3257
Author(s) : Parnas H , Slutsky I , Rashkovan G , Silman I , Wess J , Parnas I
Ref : Journal of Neurophysiology , 93 :3257 , 2005
Abstract : The role of presynaptic muscarinic autoreceptors in the initiation of phasic acetylcholine (ACh) release at frog and mouse neuromuscular junctions was studied by measuring the dependency of the amount (m) of ACh release on the level of presynaptic depolarization. Addition of methoctramine (a blocker of M2 muscarinic receptors), or of acetylcholinesterase (AChE), increased release in a voltage-dependent manner; enhancement of release declined as the depolarizing pulse amplitude increased. In frogs and wild-type mice the slope of log m/log pulse amplitude (PA) was reduced from about 7 in the control to about 4 in the presence of methoctramine or AChE. In M2 muscarinic receptor knockout mice, the slope of log m/log PA was much smaller (about 4) and was not further reduced by addition of either methoctramine or AChE. The effect of a brief (0.1 ms), but strong (-1.2 microA) depolarizing prepulse on the dependency of m on PA was also studied. The depolarizing prepulse had effects similar to those of methoctramine and AChE. In particular, it enhanced release of test pulses in a voltage-dependent manner and reduced the slope of log m/log PA from about 7 to about 4. Methoctramine + AChE occluded the prepulse effects. In knockout mice, the depolarizing prepulse had no effects. The cumulative results suggest that initiation of phasic ACh release is achieved by depolarization-mediated relief of a tonic block imposed by presynaptic M2 muscarinic receptors.
ESTHER : Parnas_2005_J.Neurophysiol_93_3257
PubMedSearch : Parnas_2005_J.Neurophysiol_93_3257
PubMedID: 15703226

Title : Acetylcholinesterase: 'classical' and 'non-classical' functions and pharmacology - Silman_2005_Curr.Opin.Pharmacol_5_293
Author(s) : Silman I , Sussman JL
Ref : Curr Opin Pharmacol , 5 :293 , 2005
Abstract : The synaptic enzyme acetylcholinesterase (AChE) terminates transmission at cholinergic synapses by rapidly hydrolysing acetylcholine. It is anchored within the synaptic cleft by a highly specialized anchoring device in which catalytic subunit tetramers assemble around a polyproline II helix. AChE is the target of nerve agents, insecticides and therapeutic drugs, in particular the first generation of anti-Alzheimer drugs. Both target-guided synthesis and structure-based drug design have been used effectively to obtain potent anticholinesterase agents. In addition, AChE is believed to play 'non-classical' roles in addition to its 'classical' role in terminating synaptic transmission (e.g. as an adhesion protein). It also accelerates assembly of Abeta into amyloid fibrils. Both of these actions involve the so-called 'peripheral' anionic site at the entrance to the active-site gorge. Novel anticholinesterases are targeted against this site, rather than against the active site at the bottom of the gorge.
ESTHER : Silman_2005_Curr.Opin.Pharmacol_5_293
PubMedSearch : Silman_2005_Curr.Opin.Pharmacol_5_293
PubMedID: 15907917

Title : Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes - Harel_2004_Nat.Struct.Mol.Biol_11_412
Author(s) : Harel M , Aharoni A , Gaidukov L , Brumshtein B , Khersonsky O , Meged R , Dvir H , Ravelli RB , McCarthy A , Toker L , Silman I , Sussman JL , Tawfik DS
Ref : Nat Struct Mol Biol , 11 :412 , 2004
Abstract : Members of the serum paraoxonase (PON) family have been identified in mammals and other vertebrates, and in invertebrates. PONs exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve agents. PON1 and PON3 reside on high-density lipoprotein (HDL, 'good cholesterol') and are involved in the prevention of atherosclerosis. We describe the first crystal structure of a PON family member, a variant of PON1 obtained by directed evolution, at a resolution of 2.2 A. PON1 is a six-bladed beta-propeller with a unique active site lid that is also involved in HDL binding. The three-dimensional structure and directed evolution studies permit a detailed description of PON1's active site and catalytic mechanism, which are reminiscent of secreted phospholipase A2, and of the routes by which PON family members diverged toward different substrate and reaction selectivities.
ESTHER : Harel_2004_Nat.Struct.Mol.Biol_11_412
PubMedSearch : Harel_2004_Nat.Struct.Mol.Biol_11_412
PubMedID: 15098021

Title : Poster (31) Natively unstructured proteins: a case study of cholinesterase-like adhesion molecules (clams) -
Author(s) : Sussman JL , Zeev-Ben-Mordehai T , Rydberg EH , Solomon A , Toker L , Botti SA , Auld VJ , Silman I
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :337 , 2004
PubMedID:

Title : Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization - Aharoni_2004_Proc.Natl.Acad.Sci.U.S.A_101_482
Author(s) : Aharoni A , Gaidukov L , Yagur S , Toker L , Silman I , Tawfik DS
Ref : Proc Natl Acad Sci U S A , 101 :482 , 2004
Abstract : Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate (OP) detoxification and in prevention of atherosclerosis. However, their structure and mechanism of action are poorly understood. PONs seem like jacks-of-all-trades, acting on a very wide range of substrates, most of which are of no physiological relevance. Family shuffling and screening lead to the first PON variants that express in a soluble and active form in Escherichia coli. We describe variants with kinetic parameters similar to those reported for PONs purified from sera and others that show dramatically increased activities. In particular, we have evolved PON1 variants with OP-hydrolyzing activities 40-fold higher than wild type and a specificity switch of >2,000-fold, producing PONs specialized for OP rather than ester hydrolysis. Analysis of the newly evolved variants provides insights into the evolutionary relationships between different family members.
ESTHER : Aharoni_2004_Proc.Natl.Acad.Sci.U.S.A_101_482
PubMedSearch : Aharoni_2004_Proc.Natl.Acad.Sci.U.S.A_101_482
PubMedID: 14695884

Title : Poster (47) Conformational plasticity of acetylcholinesterase -
Author(s) : Silman I , Greenblatt HM , Zeev-Ben-Mordehai T , Sussman JL
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :346 , 2004
PubMedID:

Title : Poster (48) Crystal structure of the tetramerization domain of acetylcholinesterase reveals a model of the AChE tetramer -
Author(s) : Harel M , Dvir H , Bon S , Liu WQ , Garbay C , Sussman JL , Massoulie J , Silman I
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :347 , 2004
PubMedID:

Title : The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix - Dvir_2004_EMBO.J_23_4394
Author(s) : Dvir H , Harel M , Bon S , Liu WQ , Vidal M , Garbay C , Sussman JL , Massoulie J , Silman I
Ref : EMBO Journal , 23 :4394 , 2004
Abstract : Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD. The WAT chains are related by an approximately 4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT-WAT and WAT-PRAD interactions. A model is proposed for the synaptic AChE(T) tetramer.
ESTHER : Dvir_2004_EMBO.J_23_4394
PubMedSearch : Dvir_2004_EMBO.J_23_4394
PubMedID: 15526038
Gene_locus related to this paper: torca-ACHE

Title : The complex of a bivalent derivative of galanthamine with torpedo acetylcholinesterase displays drastic deformation of the active-site gorge: implications for structure-based drug design - Greenblatt_2004_J.Am.Chem.Soc_126_15405
Author(s) : Greenblatt HM , Guillou C , Guenard D , Argaman A , Botti SA , Badet B , Thal C , Silman I , Sussman JL
Ref : Journal of the American Chemical Society , 126 :15405 , 2004
Abstract : Bifunctional derivatives of the alkaloid galanthamine, designed to interact with both the active site of the enzyme acetylcholinesterase (AChE) and its peripheral cation binding site, have been assayed with Torpedo californica AChE (TcAChE), and the three-dimensional structures of their complexes with the enzyme have been solved by X-ray crystallography. Differences were noted between the IC(50) values obtained for TcAChE and those for Electrophorus electricus AChE. These differences are ascribed to sequence differences in one or two residues lining the active-site gorge of the enzyme. The binding of one of the inhibitors disrupts the native conformation of one wall of the gorge, formed by the loop Trp279-Phe290. It is proposed that flexibility of this loop may permit the binding of inhibitors such as galanthamine, which are too bulky to penetrate the narrow neck of the gorge formed by Tyr121 and Phe330 as seen in the crystal structure.
ESTHER : Greenblatt_2004_J.Am.Chem.Soc_126_15405
PubMedSearch : Greenblatt_2004_J.Am.Chem.Soc_126_15405
PubMedID: 15563167
Gene_locus related to this paper: torca-ACHE

Title : Overexpression of the extracellular and cytoplasmic domains of the Drosophila adhesion protein, gliotactin. -
Author(s) : Rydberg EH , Macion R , Zeev-Ben-Mordehai T , Solomon A , Rees DM , Toker L , Botti SA , Auld VJ , Silman I , Sussman JL
Ref : Cholinergic Mechanisms, CRC Press :687 , 2004
PubMedID:

Title : Poster (74) Structural studies on torpedo californica acetylcholinesterase in complex with a substrate analogue -
Author(s) : Colletier JP , Fournier D , Greenblatt HM , Sussman JL , Zaccai G , Silman I , Weik M
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :359 , 2004
PubMedID:

Title : Poster (79) X-ray structures of TcAChE complexed with (+)-huperzine a and (-)-huperzine b: structural evidence for an active-site rearrangement -
Author(s) : Dvir H , Jiang H , Wong DM , Harel M , Chetrit M , He XC , Jin GY , Yu GL , Tang XC , Silman I , Bai DL , Sussman JL
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :362 , 2004
PubMedID:

Title : Use of the morphing graphics technique to visualize conformational differences between acetylcholinesterases from different species and inhibitor-induced conformational changes. -
Author(s) : Zeev-Ben-Mordehai T , Silman I , Sussman JL
Ref : Cholinergic Mechanisms, CRC Press :747 , 2004
PubMedID:

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

Title : Crystal structure of the tetramerization domain of acetylcholinesterase at 2. -
Author(s) : Harel M , Dvir H , Bon S , Liu WQ , Garbay C , Sussman JL , Massoulie J , Silman I
Ref : Cholinergic Mechanisms, CRC Press :183 , 2004
PubMedID:

Title : A paradigm for single nucleotide polymorphism analysis: the case of the acetylcholinesterase gene - Hasin_2004_Hum.Mutat_24_408
Author(s) : Hasin Y , Avidan N , Bercovich D , Korczyn A , Silman I , Beckmann JS , Sussman JL
Ref : Hum Mutat , 24 :408 , 2004
Abstract : Acetylcholinesterase (AChE) plays a crucial physiological role in termination of impulse transmission at cholinergic synapses through rapid hydrolysis of acetylcholine. It is a highly conserved molecule, and only a few naturally occurring genetic polymorphisms have been reported in the human gene. The goal of the present study was to make a systematic effort to identify natural single nucleotide polymorphisms (SNPs) in the human ACHE gene. To this end, the genomic coding sequences for acetylcholinesterase of 96 unrelated control individuals from three distinct ethnic groups were analyzed. A total of 13 ACHE SNPs were identified, 10 of which are newly described, and five that should produce amino acid substitutions [c.101G>A (p.Arg34Gln), c.169G>A (p.Gly57Arg), c.1031A>G (p.Glu344Gly), c.1057C>A (p.His353Asn), and c.1775C>G (p.Pro592Arg)]. Population frequencies of 11 of the 13 SNPs were established in four different populations: African Americans, Ashkenazi Jews, Sephardic Jews, and Israeli Arabs; 15 haplotypes and five ethnospecific alleles were identified. The low number of SNPs identified until now in the ACHE gene is ascribed to technical hurdles arising from the high GC content and the presence of numerous repeat sequences, and does not reflect its intrinsic heterozygosity. Among the SNPs resulting in an amino acid substitution, three are within the mature protein, mapping on its external surface: they are thus unlikely to affect its catalytic properties, yet could have antigenic consequences or affect putative protein-protein interactions. Furthermore, the newly identified SNPs open the door to a study of the possible association of AChE with deleterious phenotypes-such as adverse drug responses to AChE inhibitors employed in treatment of Alzheimer patients and hypersensitivity to pesticides.
ESTHER : Hasin_2004_Hum.Mutat_24_408
PubMedSearch : Hasin_2004_Hum.Mutat_24_408
PubMedID: 15459952

Title : Poster (104) Biophysical and bioinformatic analysis of the cytoplasmic portion of gliotactin -
Author(s) : Zeev-Ben-Mordehai T , Rydberg EH , Solomon A , Toker L , Silman I , Sussman JL
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :375 , 2004
PubMedID:

Title : New biosensors for improved detection of environmental and food contamination by anticholinesterase pesticides -
Author(s) : Toutant JP , Massoulie J , Fournier D , Marty JL , Schmid RD , Pfeiffer D , Selkirk ME , Sussman JL , Silman I , Talesa V , Wodak SJ , Stojan J , Magearu V
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :233 , 2004
PubMedID:

Title : Poster (24) New biosensors for improved detection of environmental contamination by anticholinesterase pesticides -
Author(s) : Toutant JP , Massoulie J , Fournier D , Schmid RD , Pfeiffer D , Selkirk ME , Sussman JL , Silman I , Talesa V , Wodak SJ
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :333 , 2004
PubMedID:

Title : The intracellular domain of the Drosophila cholinesterase-like neural adhesion protein, gliotactin, is natively unfolded - Zeev-Ben-Mordehai_2003_Proteins_53_758
Author(s) : Zeev-Ben-Mordehai T , Rydberg EH , Solomon A , Toker L , Auld VJ , Silman I , Botti SA , Sussman JL
Ref : Proteins , 53 :758 , 2003
Abstract : Drosophila gliotactin (Gli) is a 109-kDa transmembrane, cholinesterase-like adhesion molecule (CLAM), expressed in peripheral glia, that is crucial for formation of the blood-nerve barrier. The intracellular portion (Gli-cyt) was cloned and expressed in the cytosolic fraction of Escherichia coli BLR(DE3) at 45 mg/L and purified by Ni-NTA (nitrilotriacetic acid) chromatography. Although migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), under denaturing conditions, was unusually slow, molecular weight determination by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) confirmed that the product was consistent with its theoretical size. Gel filtration chromatography yielded an anomalously large Stokes radius, suggesting a fully unfolded conformation. Circular dichroism (CD) spectroscopy demonstrated that Gli-cyt was >50% unfolded, further suggesting a nonglobular conformation. Finally, 1D-(1)H NMR conclusively demonstrated that Gli-cyt possesses an extended unfolded structure. In addition, Gli-cyt was shown to possess charge and hydrophobic properties characteristic of natively unfolded proteins (i.e., proteins that, when purified, are intrinsically disordered under physiologic conditions in vitro).
ESTHER : Zeev-Ben-Mordehai_2003_Proteins_53_758
PubMedSearch : Zeev-Ben-Mordehai_2003_Proteins_53_758
PubMedID: 14579366

Title : How does huperzine A enter and leave the binding gorge of acetylcholinesterase? Steered molecular dynamics simulations - Xu_2003_J.Am.Chem.Soc_125_11340
Author(s) : Xu Y , Shen J , Luo X , Silman I , Sussman JL , Chen K , Jiang H
Ref : Journal of the American Chemical Society , 125 :11340 , 2003
Abstract : The entering and leaving processes of Huperzine A (HupA) binding with the long active-site gorge of Torpedo californica acetylcholinesterase (TcAChE) have been investigated by using steered molecular dynamics simulations. The analysis of the force required along the pathway shows that it is easier for HupA to bind to the active site of AChE than to disassociate from it, which for the first time interprets at the atomic level the previous experimental result that unbinding process of HupA is much slower than its binding process to AChE. The direct hydrogen bonds, water bridges, and hydrophobic interactions were analyzed during two steered molecular dynamics (SMD) simulations. Break of the direct hydrogen bond needs a great pulling force. The steric hindrance of bottleneck might be the most important factor to produce the maximal rupture force for HupA to leave the binding site but it has a little effect on the binding process of HupA with AChE. Residue Asp72 forms a lot of water bridges with HupA leaving and entering the AChE binding gorge, acting as a clamp to take out HupA from or put HupA into the active site. The flip of the peptide bond between Gly117 and Gly118 has been detected during both the conventional MD and SMD simulations. The simulation results indicate that this flip phenomenon could be an intrinsic property of AChE and the Gly117-Gly118 peptide bond in both HupA bound and unbound AChE structures tends to adopt the native enzyme structure. At last, in a vacuum the rupture force is increased up to 1500 pN while in water solution the greatest rupture force is about 800 pN, which means water molecules in the binding gorge act as lubricant to facilitate HupA entering or leaving the binding gorge.
ESTHER : Xu_2003_J.Am.Chem.Soc_125_11340
PubMedSearch : Xu_2003_J.Am.Chem.Soc_125_11340
PubMedID: 16220957

Title : Acetylcholinesterase Complexed with Bivalent Ligands Related to Huperzine A: Experimental Evidence for Species-Dependent Protein-Ligand Complementarity - Wong_2003_J.Am.Chem.Soc_125_363
Author(s) : Wong DM , Greenblatt HM , Dvir H , Carlier PR , Han YF , Pang YP , Silman I , Sussman JL
Ref : J Am Chem Soc , 125 :363 , 2003
Abstract : Acetylcholinesterase (AChE) inhibitors improve the cognitive abilities of Alzheimer patients. (-)-Huperzine A [(-)-HupA], an alkaloid isolated from the club moss, Huperzia serrata, is one such inhibitor, but the search for more potent and selective drugs continues. Recently, alkylene-linked dimers of 5-amino-5,6,7,8-tetrahydroquinolinone (hupyridone, 1a), a fragment of HupA, were shown to serve as more potent inhibitors of AChE than (-)-HupA and monomeric 1a. We soaked two such dimers, (S,S)-(-)-bis(10)-hupyridone [(S,S)-(-)-2a] and (S,S)-(-)-bis(12)-hupyridone [(S,S)-(-)-2b] containing, respectively, 10 and 12 methylenes in the spacer, into trigonal TcAChE crystals, and solved the X-ray structures of the resulting complexes using the difference Fourier technique, both to 2.15 A resolution. The structures revealed one HupA-like 1a unit bound to the "anionic" subsite of the active-site, near the bottom of the active-site gorge, adjacent to Trp84, as seen for the TcAChE/(-)-HupA complex, and the second 1a unit near Trp279 in the "peripheral" anionic site at the top of the gorge, both bivalent molecules thus spanning the active-site gorge. The results confirm that the increased affinity of the dimeric HupA analogues for AChE is conferred by binding to the two "anionic" sites of the enzyme. Inhibition data show that (-)-2a binds to TcAChE approximately 6-7- and > 170-fold more tightly than (-)-2b and (-)-HupA, respectively. In contrast, previous data for rat AChE show that (-)-2b binds approximately 3- and approximately 2-fold more tightly than (-)-2a and (-)-HupA, respectively. Structural comparison of TcAChE with rat AChE, as represented by the closely related mouse AChE structure (1maa.pdb), reveals a narrower gorge for rat AChE, a perpendicular alignment of the Tyr337 ring to the gorge axis, and its conformational rigidity, as a result of hydrogen bonding between its hydroxyl group and that of Tyr341, relative to TcAChE Phe330. These structural differences in the active-site gorge explain the switch in inhibitory potency of (-)-2a and 2b and the larger dimer/(-)-HupA potency ratios observed for TcAChE relative to rat AChE. The results offer new insights into factors affecting protein-ligand complementarity within the gorge and should assist the further development of improved AChE inhibitors.
ESTHER : Wong_2003_J.Am.Chem.Soc_125_363
PubMedSearch : Wong_2003_J.Am.Chem.Soc_125_363
PubMedID: 12517147
Gene_locus related to this paper: torca-ACHE

Title : Acetylcholinesterase in motion: visualizing conformational changes in crystal structures by a morphing procedure - Zeev-Ben-Mordehai_2003_Biopolymers_68_395
Author(s) : Zeev-Ben-Mordehai T , Silman I , Sussman JL
Ref : Biopolymers , 68 :395 , 2003
Abstract : In order to visualize and appreciate conformational changes between homologous three-dimensional (3D) protein structures or protein/inhibitor complexes, we have developed a user-friendly morphing procedure. It enabled us to detect coordinated conformational changes not easily discernible by analytic methods or by comparison of static images. This procedure was applied to comparison of native Torpedo californica acetylcholinesterase and of complexes with reversible inhibitors and conjugates with covalent inhibitors. It was likewise shown to be valuable for the visualization of conformational differences between acetylcholinesterases from different species. The procedure involves generation, in Cartesian space, of 25 interpolated intermediate structures between the initial and final 3D structures, which then serve as the individual frames in a QuickTime movie.
ESTHER : Zeev-Ben-Mordehai_2003_Biopolymers_68_395
PubMedSearch : Zeev-Ben-Mordehai_2003_Biopolymers_68_395
PubMedID: 12601798

Title : Stabilization of a metastable state of Torpedo californica acetylcholinesterase by chemical chaperones - Millard_2003_Protein.Sci_12_2337
Author(s) : Millard CB , Shnyrov VL , Newstead S , Shin I , Roth E , Silman I , Weiner L
Ref : Protein Science , 12 :2337 , 2003
Abstract : Chemical modification of Torpedo californica acetylcholinesterase by the natural thiosulfinate allicin produces an inactive enzyme through reaction with the buried cysteine Cys 231. Optical spectroscopy shows that the modified enzyme is "native-like," and inactivation can be reversed by exposure to reduced glutathione. The allicin-modified enzyme is, however, metastable, and is converted spontaneously and irreversibly, at room temperature, with t(1/2) approximately 100 min, to a stable, partially unfolded state with the physicochemical characteristics of a molten globule. Osmolytes, including trimethylamine-N-oxide, glycerol, and sucrose, and the divalent cations, Ca(2+), Mg(2+), and Mn(2+) can prevent this transition of the native-like state for >24 h at room temperature. Trimethylamine-N-oxide and Mg(2+) can also stabilize the native enzyme, with only slight inactivation being observed over several hours at 39 degrees C, whereas in their absence it is totally inactivated within 5 min. The stabilizing effects of the osmolytes can be explained by their differential interaction with the native and native-like states, resulting in a shift of equilibrium toward the native state. The stabilizing effects of the divalent cations can be ascribed to direct stabilization of the native state, as supported by differential scanning calorimetry.
ESTHER : Millard_2003_Protein.Sci_12_2337
PubMedSearch : Millard_2003_Protein.Sci_12_2337
PubMedID: 14500892

Title : Acetylcholinesterase: a multifaceted target for structure-based drug design of anticholinesterase agents for the treatment of Alzheimer's disease - Greenblatt_2003_J.Mol.Neurosci_20_369
Author(s) : Greenblatt HM , Dvir H , Silman I , Sussman JL
Ref : Journal of Molecular Neuroscience , 20 :369 , 2003
Abstract : The structure of Torpedo californica acetylcholinesterase is examined in complex with several inhibitors that are either in use or under development for treating Alzheimer's disease. The noncovalent inhibitors vary greatly in their structures and bind to different sites of the enzyme, offering many different starting points for future drug design.
ESTHER : Greenblatt_2003_J.Mol.Neurosci_20_369
PubMedSearch : Greenblatt_2003_J.Mol.Neurosci_20_369
PubMedID: 14501022

Title : Evidence for the formation of disulfide radicals in protein crystals upon X-ray irradiation - Weik_2002_J.Synchrotron.Radiat_9_342
Author(s) : Weik M , Berges J , Raves ML , Gros P , McSweeney S , Silman I , Sussman JL , Houee-Levin C , Ravelli RB
Ref : J Synchrotron Radiat , 9 :342 , 2002
Abstract : Irradiation of proteins with intense X-ray radiation produced by third-generation synchrotron sources generates specific structural and chemical alterations, including breakage of disulfide bonds and decarboxylation. In this paper, disulfide bond lengths in irradiated crystals of the enzyme Torpedo californica acetylcholinesterase are examined based on quantum simulations and on experimental data published previously. The experimental data suggest that one disulfide bond elongates by approximately 0.7 A upon X-ray irradiation as seen in a series of nine data sets collected on a single crystal. Simulation of the same bond suggests elongation by a similar value if a disulfide-radical anion is formed by trapping an electron. The absorption spectrum of a crystal irradiated under similar conditions shows a peak at approximately 400 nm, which in aqueous solution has been attributed to disulfide radicals. The results suggest that the formation of disulfide radicals in protein crystals owing to X-ray irradiation can be observed experimentally, both by structural means and by absorption spectroscopy.
ESTHER : Weik_2002_J.Synchrotron.Radiat_9_342
PubMedSearch : Weik_2002_J.Synchrotron.Radiat_9_342
PubMedID: 12409620

Title : Kinetic and structural studies on the interaction of cholinesterases with the anti-Alzheimer drug rivastigmine - Bar-On_2002_Biochemistry_41_3555
Author(s) : Bar-On P , Millard CB , Harel M , Dvir H , Enz A , Sussman JL , Silman I
Ref : Biochemistry , 41 :3555 , 2002
Abstract : Rivastigmine, a carbamate inhibitor of acetylcholinesterase, is already in use for treatment of Alzheimer's disease under the trade name of Exelon. Rivastigmine carbamylates Torpedo californica acetylcholinesterase very slowly (k(i) = 2.0 M(-1) min(-1)), whereas the bimolecular rate constant for inhibition of human acetylcholinesterase is >1600-fold higher (k(i) = 3300 M(-1) min(-1)). For human butyrylcholinesterase and for Drosophila melanogaster acetylcholinesterase, carbamylation is even more rapid (k(i) = 9 x 10(4) and 5 x 10(5) M(-1) min(-1), respectively). Spontaneous reactivation of all four conjugates is very slow, with <10% reactivation being observed for the Torpedo enzyme after 48 h. The crystal structure of the conjugate of rivastigmine with Torpedo acetylcholinesterase was determined to 2.2 A resolution. It revealed that the carbamyl moiety is covalently linked to the active-site serine, with the leaving group, (-)-S-3-[1-(dimethylamino)ethyl]phenol, being retained in the "anionic" site. A significant movement of the active-site histidine (H440) away from its normal hydrogen-bonded partner, E327, was observed, resulting in disruption of the catalytic triad. This movement may provide an explanation for the unusually slow kinetics of reactivation.
ESTHER : Bar-On_2002_Biochemistry_41_3555
PubMedSearch : Bar-On_2002_Biochemistry_41_3555
PubMedID: 11888271
Gene_locus related to this paper: torca-ACHE

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 : X-ray Structures of Torpedo californica Acetylcholinesterase Complexed with (+)-Huperzine A and (-)-Huperzine B: Structural Evidence for an Active Site Rearrangement - Dvir_2002_Biochemistry_41_10810
Author(s) : Dvir H , Jiang H , Wong DM , Harel M , Chetrit M , He XC , Jin GY , Yu GL , Tang XC , Silman I , Bai DL , Sussman JL
Ref : Biochemistry , 41 :10810 , 2002
Abstract : Kinetic and structural data are presented on the interaction with Torpedo californica acetylcholinesterase (TcAChE) of (+)-huperzine A, a synthetic enantiomer of the anti-Alzheimer drug, (-)-huperzine A, and of its natural homologue (-)-huperzine B. (+)-Huperzine A and (-)-huperzine B bind to the enzyme with dissociation constants of 4.30 and 0.33 microM, respectively, compared to 0.18 microM for (-)-huperzine A. The X-ray structures of the complexes of (+)-huperzine A and (-)-huperzine B with TcAChE were determined to 2.1 and 2.35 A resolution, respectively, and compared to the previously determined structure of the (-)-huperzine A complex. All three interact with the "anionic" subsite of the active site, primarily through pi-pi stacking and through van der Waals or C-H.pi interactions with Trp84 and Phe330. Since their alpha-pyridone moieties are responsible for their key interactions with the active site via hydrogen bonding, and possibly via C-H.pi interactions, all three maintain similar positions and orientations with respect to it. The carbonyl oxygens of all three appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between Gly117 and Gly118 to undergo a peptide flip. As a consequence, the position of the main chain nitrogen of Gly118 in the "oxyanion" hole in the native enzyme becomes occupied by the carbonyl of Gly117. Furthermore, the flipped conformation is stabilized by hydrogen bonding of Gly117O to Gly119N and Ala201N, the other two functional elements of the three-pronged "oxyanion hole" characteristic of cholinesterases. All three inhibitors thus would be expected to abolish hydrolysis of all ester substrates, whether charged or neutral.
ESTHER : Dvir_2002_Biochemistry_41_10810
PubMedSearch : Dvir_2002_Biochemistry_41_10810
PubMedID: 12196020
Gene_locus related to this paper: torca-ACHE

Title : Structure of a complex of the potent and specific inhibitor BW284C51 with Torpedo californica acetylcholinesterase - Felder_2002_Acta.Crystallogr.D.Biol.Crystallogr_58_1765
Author(s) : Felder CE , Harel M , Silman I , Sussman JL
Ref : Acta Crystallographica D Biol Crystallogr , 58 :1765 , 2002
Abstract : The X-ray crystal structure of Torpedo californica acetylcholinesterase (TcAChE) complexed with BW284C51 [CO[-CH(2)CH(2)-pC(6)H(4)-N(CH(3))(2)(CH(2)-CH=CH(2))](2)] is described and compared with the complexes of two other active-site gorge-spanning inhibitors, decamethonium and E2020. The inhibitor was soaked into TcAChE crystals in the trigonal space group P3(1)21, yielding a complex which diffracted to 2.85 A resolution. The structure was refined to an R factor of 19.0% and an R(free) of 23.4%; the final model contains the protein, inhibitor, 132 water molecules and three carbohydrate moieties. BW284C51 binds similarly to decamethonium and E2020, with its two phenyl and quaternary amino end-groups complexed to Trp84 in the catalytic site and to Trp279 in the peripheral binding site, and its central carbonyl group hydrogen bonded very weakly to Tyr121. Possible reasons for decamethonium's weaker binding are considered. The relative strength of binding of bisquaternary inhibitors to acetylcholinesterase and the effect of several mutations of the enzyme are discussed in the context of the respective X-ray structures of their complexes with the enzyme.
ESTHER : Felder_2002_Acta.Crystallogr.D.Biol.Crystallogr_58_1765
PubMedSearch : Felder_2002_Acta.Crystallogr.D.Biol.Crystallogr_58_1765
PubMedID: 12351819
Gene_locus related to this paper: torca-ACHE

Title : A neutral molecule in a cation-binding site: specific binding of a PEG-SH to acetylcholinesterase from Torpedo californica - Koellner_2002_J.Mol.Biol_320_721
Author(s) : Koellner G , Steiner T , Millard CB , Silman I , Sussman JL
Ref : Journal of Molecular Biology , 320 :721 , 2002
Abstract : The crystal structure of acetylcholinesterase from Torpedo californica complexed with the uncharged inhibitor, PEG-SH-350 (containing mainly heptameric polyethylene glycol with a terminal thiol group) is determined at 2.3 A resolution. This is an untypical acetylcholinesterase inhibitor, since it lacks the cationic moiety typical of the substrate (acetylcholine). In the crystal structure, the elongated ligand extends along the whole of the deep and narrow active-site gorge, with the terminal thiol group bound near the bottom, close to the catalytic site. Unexpectedly, the cation-binding site (formed by the faces of aromatic side-chains) is occupied by CH(2) groups of the inhibitor, which are engaged in C-H...pi interactions that structurally mimic the cation-pi interactions made by the choline moiety of acetylcholine. In addition, the PEG-SH molecule makes numerous other weak but specific interactions of the C-H...O and C-H...pi types.
ESTHER : Koellner_2002_J.Mol.Biol_320_721
PubMedSearch : Koellner_2002_J.Mol.Biol_320_721
PubMedID: 12095250
Gene_locus related to this paper: torca-ACHE

Title : Thermal denaturation of Bungarus fasciatus acetylcholinesterase: Is aggregation a driving force in protein unfolding? - Shin_2002_Protein.Sci_11_2022
Author(s) : Shin I , Wachtel E , Roth E , Bon C , Silman I , Weiner L
Ref : Protein Science , 11 :2022 , 2002
Abstract : A monomeric form of acetylcholinesterase from the venom of Bungarus fasciatus is converted to a partially unfolded molten globule species by thermal inactivation, and subsequently aggregates rapidly. To separate the kinetics of unfolding from those of aggregation, single molecules of the monomeric enzyme were encapsulated in reverse micelles of Brij 30 in 2,2,4-trimethylpentane, or in large unilamellar vesicles of egg lecithin/cholesterol at various protein/micelle (vesicle) ratios. The first-order rate constant for thermal inactivation at 45 degrees C, of single molecules entrapped within the reverse micelles (0.031 min(-1)), was higher than in aqueous solution (0.007 min(-1)) or in the presence of normal micelles (0.020 min(-1)). This clearly shows that aggregation does not provide the driving force for thermal inactivation of BfAChE. Within the large unilamellar vesicles, at average protein/vesicle ratios of 1:1 and 10:1, the first-order rate constants for thermal inactivation of the encapsulated monomeric acetylcholinesterase, at 53 degrees C, were 0.317 and 0.342 min(-1), respectively. A crosslinking technique, utilizing the photosensitive probe, hypericin, showed that thermal denaturation produces a distribution of species ranging from dimers through to large aggregates. Consequently, at a protein/vesicle ratio of 10:1, aggregation can occur upon thermal denaturation. Thus, these experiments also demonstrate that aggregation does not drive the thermal unfolding of Bungarus fasciatus acetylcholinesterase. Our experimental approach also permitted monitoring of recovery of enzymic activity after thermal denaturation in the absence of a competing aggregation process. Whereas no detectable recovery of enzymic activity could be observed in aqueous solution, up to 23% activity could be obtained for enzyme sequestered in the reverse micelles.
ESTHER : Shin_2002_Protein.Sci_11_2022
PubMedSearch : Shin_2002_Protein.Sci_11_2022
PubMedID: 12142456

Title : A Structural Motif of Acetylcholinesterase That Promotes Amyloid beta-Peptide Fibril Formation - De Ferrari_2001_Biochemistry_40_10447
Author(s) : De Ferrari GV , Canales MA , Shin I , Weiner L , Silman I , Inestrosa NC
Ref : Biochemistry , 40 :10447 , 2001
Abstract : Acetylcholinesterase (AChE) has been found to be associated with the core of senile plaques. We have shown that AChE interacts with the amyloid beta-peptide (Abeta) and promotes amyloid fibril formation by a hydrophobic environment close to the peripheral anionic binding site (PAS) of the enzyme. Here we present evidence for the structural motif of AChE involved in this interaction. First, we modeled the docking of Abeta onto the structure of Torpedo californica AChE, and identified four potential sites for AChE-Abeta complex formation. One of these, Site I, spans a major hydrophobic sequence exposed on the surface of AChE, which had been previously shown to interact with liposomes [Shin et al. (1996) Protein Sci. 5, 42-51]. Second, we examined several AChE-derived peptides and found that a synthetic 35-residue peptide corresponding to the above hydrophobic sequence was able to promote amyloid formation. We also studied the ability to promote amyloid formation of two synthetic 24-residue peptides derived from the sequence of a Omega-loop, which has been suggested as an AChE-Abeta interacting motif. Kinetic analyses indicate that only the 35-residue hydrophobic peptide mimics the effect of intact AChE on amyloid formation. Moreover, RP-HPLC analysis revealed that the 35-residue peptide was incorporated into the growing Abeta-fibrils. Finally, fluorescence binding studies showed that this peptide binds Abeta with a K(d) = 184 microM, independent of salt concentration, indicating that the interaction is primarily hydrophobic. Our results indicate that the homologous human AChE motif is capable of accelerating Abeta fibrillogenesis.
ESTHER : De Ferrari_2001_Biochemistry_40_10447
PubMedSearch : De Ferrari_2001_Biochemistry_40_10447
PubMedID: 11523986

Title : Histochemical method for characterization of enzyme crystals: application to crystals of Torpedo californica acetylcholinesterase - Nicolas_2001_Acta.Crystallogr.D.Biol.Crystallogr_57_1348
Author(s) : Nicolas A , Ferron F , Toker L , Sussman JL , Silman I
Ref : Acta Crystallographica D Biol Crystallogr , 57 :1348 , 2001
Abstract : Histochemical methods are employed to detect and localize a wide range of enzymes. Even though protein crystallographers do not commonly use this technique, the extensively used colorimetric reaction of Karnovsky was successfully adapted for easy and quick identification of acetylcholinesterase crystals. The method relies on the reduction of ferricyanide to ferrocyanide by thiocholine, released from acetylthiocholine by enzymatic hydrolysis, followed by formation of a cupric ferrocyanide precipitate, and allows rapid differentiation between salt and enzyme crystals and between native and inhibited crystals of the enzyme.
ESTHER : Nicolas_2001_Acta.Crystallogr.D.Biol.Crystallogr_57_1348
PubMedSearch : Nicolas_2001_Acta.Crystallogr.D.Biol.Crystallogr_57_1348
PubMedID: 11526341

Title : A structure-based design approach to the development of novel, reversible AChE inhibitors - Doucet-Personeni_2001_J.Med.Chem_44_3203
Author(s) : Doucet-Personeni C , Bentley PD , Fletcher RJ , Kinkaid A , Kryger G , Pirard B , Taylor A , Taylor R , Taylor J , Viner R , Silman I , Sussman JL , Greenblatt HM , Lewis T
Ref : Journal of Medicinal Chemistry , 44 :3203 , 2001
Abstract : Chimeras of tacrine and m-(N,N,N-Trimethylammonio)trifluoroacetophenone (1) were designed as novel, reversible inhibitors of acetylcholinesterase. On the basis of the X-ray structure of the apoenzyme, a molecular modeling study determined the favored attachment positions on the 4-aminoquinoline ring (position 3 and the 4-amino nitrogen) and the favored lengths of a polymethylene link between the two moieties (respectively 5-6 and 4-5 sp(3) atoms). Seven compounds matching these criteria were synthesized, and their inhibitory potencies were determined to be in the low nanomolar range. Activity data for close analogues lacking some of the postulated key features showed that our predictions were correct. In addition, a subsequent crystal structure of acetylcholinesterase complexed with the most active compound 27 was in good agreement with our model. The design strategy is therefore validated and can now be developed further.
ESTHER : Doucet-Personeni_2001_J.Med.Chem_44_3203
PubMedSearch : Doucet-Personeni_2001_J.Med.Chem_44_3203
PubMedID: 11563919
Gene_locus related to this paper: torca-ACHE

Title : Specific protein dynamics near the solvent glass transition assayed by radiation-induced structural changes - Weik_2001_Protein.Sci_10_1953
Author(s) : Weik M , Ravelli RB , Silman I , Sussman JL , Gros P , Kroon J
Ref : Protein Science , 10 :1953 , 2001
Abstract : The nature of the dynamical coupling between a protein and its surrounding solvent is an important, yet open issue. Here we used temperature-dependent protein crystallography to study structural alterations that arise in the enzyme acetylcholinesterase upon X-ray irradiation at two temperatures: below and above the glass transition of the crystal solvent. A buried disulfide bond, a buried cysteine, and solvent exposed methionine residues show drastically increased radiation damage at 155 K, in comparison to 100 K. Additionally, the irradiation-induced unit cell volume increase is linear at 100 K, but not at 155 K, which is attributed to the increased solvent mobility at 155 K. Most importantly, we observed conformational changes in the catalytic triad at the active site at 155 K but not at 100 K. These changes lead to an inactive catalytic triad conformation and represent, therefore, the observation of radiation-inactivation of an enzyme at the atomic level. Our results show that at 155 K, the protein has acquired--at least locally--sufficient conformational flexibility to adapt to irradiation-induced alterations in the conformational energy landscape. The increased protein flexibility may be a direct consequence of the solvent glass transition, which expresses as dynamical changes in the enzyme's environment. Our results reveal the importance of protein and solvent dynamics in specific radiation damage to biological macromolecules, which in turn can serve as a tool to study protein flexibility and its relation to changes in a protein's environment.
ESTHER : Weik_2001_Protein.Sci_10_1953
PubMedSearch : Weik_2001_Protein.Sci_10_1953
PubMedID: 11567086

Title : Presynaptic M(2) muscarinic receptors are involved in controlling the kinetics of ACh release at the frog neuromuscular junction - Slutsky_2001_J.Physiol_536_717
Author(s) : Slutsky I , Silman I , Parnas I , Parnas H
Ref : The Journal of Physiology , 536 :717 , 2001
Abstract : 1. Macropatch recording was used to study release of acetylcholine in the frog neuromuscular junction evoked by either direct local depolarization or by an action potential. 2. The quantal content was established by directly counting the released quanta. The time course of release was obtained by constructing synaptic delay histograms. 3. Perfusion of the neuromuscular junction with methoctramine, a selective M(2)/M(4) muscarinic antagonist, increased the quantal content and slowed the exponential decay of the synaptic delay histograms. Addition of the agonist muscarine reversed these effects. 4. Addition of acetylcholinesterase prolonged the decay of the delay histogram, and muscarine reversed this effect. 5. Methoctramine slowed the rise time of the postsynaptic current produced by axon stimulation without affecting either the excitatory nerve terminal current or the presynaptic Ca(2+) current. 6. These results show that presynaptic M(2) muscarinic receptors are involved in the process which terminates evoked ACh release.
ESTHER : Slutsky_2001_J.Physiol_536_717
PubMedSearch : Slutsky_2001_J.Physiol_536_717
PubMedID: 11691867

Title : Active-site gorge and buried water molecules in crystal structures of acetylcholinesterase from Torpedo californica - Koellner_2000_J.Mol.Biol_296_713
Author(s) : Koellner G , Kryger G , Millard CB , Silman I , Sussman JL , Steiner T
Ref : Journal of Molecular Biology , 296 :713 , 2000
Abstract : Buried water molecules and the water molecules in the active-site gorge are analyzed for five crystal structures of acetylcholinesterase from Torpedo californica in the resolution range 2.2-2.5 A (native enzyme, and four inhibitor complexes). A total of 45 buried hydration sites are identified, which are populated with between 36 and 41 water molecules. About half of the buried water is located in a distinct region neighboring the active-site gorge. Most of the buried water molecules are very well conserved among the five structures, and have low displacement parameters, B, of magnitudes similar to those of the main-chain atoms of the central beta-sheet structure. The active-site gorge of the native enzyme is filled with over 20 water molecules, which have poor hydrogen-bond coordination with an average of 2.9 polar contacts per water molecule. Upon ligand binding, distinct groups of these water molecules are displaced, whereas the others remain in positions similar to those that they occupy in the native enzyme. Possible roles of the buried water molecules are discussed, including their possible action as a lubricant to allow large-amplitude fluctuations of the loop structures forming the gorge wall. Such fluctuations are required to facilitate traffic of substrate, products and water molecules to and from the active-site. Because of their poor coordination, the gorge water molecules can be considered as "activated" as compared to bulk water. This should allow their easy displacement by incoming substrate. The relatively loose packing of the gorge water molecules leaves numerous small voids, and more efficient space-filling by substrates and inhibitors may be a major driving force of ligand binding.
ESTHER : Koellner_2000_J.Mol.Biol_296_713
PubMedSearch : Koellner_2000_J.Mol.Biol_296_713
PubMedID: 10669619

Title : Specific chemical and structural damage to proteins produced by synchrotron radiation - Weik_2000_Proc.Natl.Acad.Sci.U.S.A_97_623
Author(s) : Weik M , Ravelli RB , Kryger G , McSweeney S , Raves ML , Harel M , Gros P , Silman I , Kroon J , Sussman JL
Ref : Proceedings of the National Academy of Sciences of the United States of America , 97 :623 , 2000
Abstract : Radiation damage is an inherent problem in x-ray crystallography. It usually is presumed to be nonspecific and manifested as a gradual decay in the overall quality of data obtained for a given crystal as data collection proceeds. Based on third-generation synchrotron x-ray data, collected at cryogenic temperatures, we show for the enzymes Torpedo californica acetylcholinesterase and hen egg white lysozyme that synchrotron radiation also can cause highly specific damage. Disulfide bridges break, and carboxyl groups of acidic residues lose their definition. Highly exposed carboxyls, and those in the active site of both enzymes, appear particularly susceptible. The catalytic triad residue, His-440, in acetylcholinesterase, also appears to be much more sensitive to radiation damage than other histidine residues. Our findings have direct practical implications for routine x-ray data collection at high-energy synchrotron sources. Furthermore, they provide a direct approach for studying the radiation chemistry of proteins and nucleic acids at a detailed, structural level and also may yield information concerning putative "weak links" in a given biological macromolecule, which may be of structural and functional significance.
ESTHER : Weik_2000_Proc.Natl.Acad.Sci.U.S.A_97_623
PubMedSearch : Weik_2000_Proc.Natl.Acad.Sci.U.S.A_97_623
PubMedID: 10639129
Gene_locus related to this paper: torca-ACHE

Title : Inactivation studies of acetylcholinesterase with phenylmethylsulfonyl fluoride - Kraut_2000_Mol.Pharmacol_57_1243
Author(s) : Kraut D , Goff H , Pai RK , Hosea NA , Silman I , Sussman JL , Taylor P , Voet JG
Ref : Molecular Pharmacology , 57 :1243 , 2000
Abstract : Acetylcholinesterase (AChE), a serine hydrolase, is potentially susceptible to inactivation by phenylmethylsulfonyl fluoride (PMSF) and benzenesulfonyl fluoride (BSF). Although BSF inhibits both mouse and Torpedo californica AChE, PMSF does not react measurably with the T. californica enzyme. To understand the residue changes responsible for the change in reactivity, we studied the inactivation of wild-type T. californica and mouse AChE and mutants of both by BSF and PMSF both in the presence and absence of substrate. The enzymes investigated were wild-type mouse AChE, wild-type T. californica AChE, wild-type mouse butyrylcholinesterase, mouse Y330F, Y330A, F288L, and F290I, and the double mutant T. californica F288L/F290V (all mutants given T. californica numbering). Inactivation rate constants for T. californica AChE confirmed previous reports that this enzyme is not inactivated by PMSF. Wild-type mouse AChE and mouse mutants Y330F and Y330A all had similar inactivation rate constants with PMSF, implying that the difference between mouse and T. californica AChE at position 330 is not responsible for their differing PMSF sensitivities. In addition, butyrylcholinesterase and mouse AChE mutants F288L and F290I had increased rate constants ( approximately 14 fold) over those of wild-type mouse AChE, indicating that these residues may be responsible for the increased sensitivity to inactivation by PMSF of butyrylcholinesterase. The double mutant T. californica AChE F288L/F290V had a rate constant nearly identical with the rate constant for the F288L and F290I mouse mutant AChEs, representing an increase of approximately 4000-fold over the T. californica wild-type enzyme. It remains unclear why these two positions have more importance for T. californica AChE than for mouse AChE.
ESTHER : Kraut_2000_Mol.Pharmacol_57_1243
PubMedSearch : Kraut_2000_Mol.Pharmacol_57_1243
PubMedID: 10825396

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 : Structures of recombinant native and E202Q mutant human acetylcholinesterase complexed with the snake-venom toxin fasciculin-II - Kryger_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1385
Author(s) : Kryger G , Harel M , Giles K , Toker L , Velan B , Lazar A , Kronman C , Barak D , Ariel N , Shafferman A , Silman I , Sussman JL
Ref : Acta Crystallographica D Biol Crystallogr , 56 :1385 , 2000
Abstract : Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a 'three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 A resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 A resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.
ESTHER : Kryger_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1385
PubMedSearch : Kryger_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1385
PubMedID: 11053835
Gene_locus related to this paper: human-ACHE

Title : A modular treatment of molecular traffic through the active site of cholinesterase - Botti_1999_Biophys.J_77_2430
Author(s) : Botti SA , Felder CE , Lifson S , Sussman JL , Silman I
Ref : Biophysical Journal , 77 :2430 , 1999
Abstract : We present a model for the molecular traffic of ligands, substrates, and products through the active site of cholinesterases (ChEs). First, we describe a common treatment of the diffusion to a buried active site of cationic and neutral species. We then explain the specificity of ChEs for cationic ligands and substrates by introducing two additional components to this common treatment. The first module is a surface trap for cationic species at the entrance to the active-site gorge that operates through local, short-range electrostatic interactions and is independent of ionic strength. The second module is an ionic-strength-dependent steering mechanism generated by long-range electrostatic interactions arising from the overall distribution of charges in ChEs. Our calculations show that diffusion of charged ligands relative to neutral isosteric analogs is enhanced approximately 10-fold by the surface trap, while electrostatic steering contributes only a 1.5- to 2-fold rate enhancement at physiological salt concentration. We model clearance of cationic products from the active-site gorge as analogous to the escape of a particle from a one-dimensional well in the presence of a linear electrostatic potential. We evaluate the potential inside the gorge and provide evidence that while contributing to the steering of cationic species toward the active site, it does not appreciably retard their clearance. This optimal fine-tuning of global and local electrostatic interactions endows ChEs with maximum catalytic efficiency and specificity for a positively charged substrate, while at the same time not hindering clearance of the positively charged products.
ESTHER : Botti_1999_Biophys.J_77_2430
PubMedSearch : Botti_1999_Biophys.J_77_2430
PubMedID: 10545346

Title : Structure of acetylcholinesterase complexed with (-)-galanthamine at 2.3 A resolution - Greenblatt_1999_FEBS.Lett_463_321
Author(s) : Greenblatt HM , Kryger G , Lewis T , Silman I , Sussman JL
Ref : FEBS Letters , 463 :321 , 1999
Abstract : (-)-Galanthamine (GAL), an alkaloid from the flower, the common snowdrop (Galanthus nivalis), shows anticholinesterase activity. This property has made GAL the target of research as to its effectiveness in the treatment of Alzheimer's disease. We have solved the X-ray crystal structure of GAL bound in the active site of Torpedo californica acetylcholinesterase (TcAChE) to 2.3 A resolution. The inhibitor binds at the base of the active site gorge of TcAChE, interacting with both the choline-binding site (Trp-84) and the acyl-binding pocket (Phe-288, Phe-290). The tertiary amine group of GAL does not interact closely with Trp-84; rather, the double bond of its cyclohexene ring stacks against the indole ring. The tertiary amine appears to make a non-conventional hydrogen bond, via its N-methyl group, to Asp-72, near the top of the gorge. The hydroxyl group of the inhibitor makes a strong hydrogen bond (2.7 A) with Glu-199. The relatively tight binding of GAL to TcAChE appears to arise from a number of moderate to weak interactions with the protein, coupled to a low entropy cost for binding due to the rigid nature of the inhibitor.
ESTHER : Greenblatt_1999_FEBS.Lett_463_321
PubMedSearch : Greenblatt_1999_FEBS.Lett_463_321
PubMedID: 10606746
Gene_locus related to this paper: torca-ACHE

Title : Effect of mutations within the peripheral anionic site on the stability of acetylcholinesterase - Morel_1999_Mol.Pharmacol_55_982
Author(s) : Morel N , Bon S , Greenblatt HM , Van Belle D , Wodak SJ , Sussman JL , Massoulie J , Silman I
Ref : Molecular Pharmacology , 55 :982 , 1999
Abstract : Torpedo acetylcholinesterase is irreversibly inactivated by modifying a buried free cysteine, Cys231, with sulfhydryl reagents. The stability of the enzyme, as monitored by measuring the rate of inactivation, was reduced by mutating a leucine, Leu282, to a smaller amino acid residue. Leu282 is located within the "peripheral" anionic site, at the entrance to the active-site gorge. Thus, loss of activity was due to the increased reactivity of Cys231. This was paralleled by an increased susceptibility to thermal denaturation, which was shown to be due to a large decrease in the activation enthalpy. Similar results were obtained when either of two other residues in contact with Leu282 in Torpedo acetylcholinesterase, Trp279 and Ser291, was replaced by an amino acid with a smaller side chain. We studied the effects of various ligands specific for either the active or peripheral sites on both thermal inactivation and on inactivation by 4,4'-dithiodipyridine. The wild-type and mutated enzymes could be either protected or sensitized. In some cases, opposite effects of the same ligand were observed for chemical modification and thermal denaturation. The mutated residues are within a conserved loop, W279-S291, at the top of the active-site gorge, that contributes to the peripheral anionic site. Theoretical analysis showed that Torpedo acetylcholinesterase consists of two structural domains, each comprising one contiguous polypeptide segment. The W279-S291 loop, located in the first domain, makes multiple contacts with the second domain across the active-site gorge. We postulate that the mutations to residues with smaller side chains destabilize the conserved loop, thus disrupting cross-gorge interactions and, ultimately, the entire structure.
ESTHER : Morel_1999_Mol.Pharmacol_55_982
PubMedSearch : Morel_1999_Mol.Pharmacol_55_982
PubMedID: 10347238

Title : Structure of acetylcholinesterase complexed with E2020 (Aricept): implications for the design of new anti-Alzheimer drugs - Kryger_1999_Structure_7_297
Author(s) : Kryger G , Silman I , Sussman JL
Ref : Structure , 7 :297 , 1999
Abstract : BACKGROUND: Several cholinesterase inhibitors are either being utilized for symptomatic treatment of Alzheimer's disease or are in advanced clinical trials. E2020, marketed as Aricept, is a member of a large family of N-benzylpiperidine-based acetylcholinesterase (AChE) inhibitors developed, synthesized and evaluated by the Eisai Company in Japan. These inhibitors were designed on the basis of QSAR studies, prior to elucidation of the three-dimensional structure of Torpedo californica AChE (TcAChE). It significantly enhances performance in animal models of cholinergic hypofunction and has a high affinity for AChE, binding to both electric eel and mouse AChE in the nanomolar range. RESULTS: Our experimental structure of the E2020-TcAChE complex pinpoints specific interactions responsible for the high affinity and selectivity demonstrated previously. It shows that E2020 has a unique orientation along the active-site gorge, extending from the anionic subsite of the active site, at the bottom, to the peripheral anionic site, at the top, via aromatic stacking interactions with conserved aromatic acid residues. E2020 does not, however, interact directly with either the catalytic triad or the 'oxyanion hole', but only indirectly via solvent molecules.
CONCLUSIONS: Our study shows, a posteriori, that the design of E2020 took advantage of several important features of the active-site gorge of AChE to produce a drug with both high affinity for AChE and a high degree of selectivity for AChE versus butyrylcholinesterase (BChE). It also delineates voids within the gorge that are not occupied by E2020 and could provide sites for potential modification of E2020 to produce drugs with improved pharmacological profiles.
ESTHER : Kryger_1999_Structure_7_297
PubMedSearch : Kryger_1999_Structure_7_297
PubMedID: 10368299
Gene_locus related to this paper: torca-ACHE

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

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

Title : Acetylcholinesterase of Schistosoma mansoni--functional correlates. Contributed in honor of Professor Hans Neurath's 90th birthday - Arnon_1999_Protein.Sci_8_2553
Author(s) : Arnon R , Silman I , Tarrab-Hazdai R
Ref : Protein Science , 8 :2553 , 1999
Abstract : Acetylcholinesterase (AChE) is an enzyme broadly distributed in many species, including parasites. It occurs in multiple molecular forms that differ in their quaternary structure and mode of anchoring to the cell surface. This review summarizes biochemical and immunological investigations carried out in our laboratories on AChE of the helmint, Schistosoma mansoni. AChE appears in S. mansoni in two principal molecular forms, both globular, with sedimentation coefficients of approximately 6.5 and 8 S. On the basis of their substrate specificity and sensitivity to inhibitors, both are "true" acetylcholinesterases. Approximately half of the AChE activity of S. mansoni is located on the outer surface of the parasite, attached to the tegumental membrane via a covalently attached glycosylphosphatidylinositol anchor. The remainder is located within the parasite, mainly associated with muscle tissue. Whereas the internal enzyme is most likely involved in termination of neurotransmission at cholinergic synapses, the role of the surface enzyme remains to be established; there are, however, indications that it is involved in signal transduction. The two forms of AChE differ in their heparin-binding properties, only the internal 8 S form of the AChE being retained on a heparin column. The two forms differ also in their immunological specificity, since they are selectively recognized by different monoclonal antibodies. Polyclonal antibodies raised against S. mansoni AChE purified by affinity chromatography are specific for the parasite AChE, reacting with both molecular forms, but do not recognize AChE from other species. They interact with the surface-localized enzyme on the intact organism, and produce almost total complement-dependent killing of the parasite. S. mansoni AChE is thus demonstrated to be a functional protein, involved in multifaceted activities, which can serve as a suitable candidate for diagnostic purposes, vaccine development, and drug design.
ESTHER : Arnon_1999_Protein.Sci_8_2553
PubMedSearch : Arnon_1999_Protein.Sci_8_2553
PubMedID: 10631970

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

Title : Acetylcholinesterase from Schistosoma mansoni: interaction of globular species with heparin - Tarrab-Hazdai_1999_Biochem.J_344 Pt 3_945
Author(s) : Tarrab-Hazdai R , Toker L , Silman I , Arnon R
Ref : Biochemical Journal , 344 Pt 3 :945 , 1999
Abstract : In the cercarial and schistosomal stages of the life cycle of the trematode Schistosoma mansoni, acetylcholinesterase occurs as two principal molecular forms (both globular), present in approximately equal amounts, with sedimentation coefficients of 6.5 S and 8 S. The 6.5 S form is solubilized by bacterial phosphatidylinositol-specific phospholipase C from intact schistosomula. It is thus located on the outer surface of the schistosomal tegument and is most probably analogous to the glycosylphosphatidylinositol-anchored G(2) form of acetylcholinesterase found in the electric organ of Torpedo, on the surface of mammalian erythrocytes, and elsewhere. Both forms are fully solubilized by the non-ionic detergent Triton X-100. Upon passing such a detergent extract over a heparin-Sepharose column, only the 8 S form was retained on the column. The bound acetylcholinesterase could be progressively eluted by increasing the salt concentration, with approx. 0.5-0.6 M NaCl being needed for complete elution. Selective inhibition experiments carried out on live parasites using the covalent acetylcholinesterase inhibitor echothiophate (phospholine), which does not penetrate the tegument, selectively inhibited the 6.5 S form, but not the 8 S form, suggesting an internal location for the latter. Monoclonal antibodies raised against S. mansoni acetylcholinesterase also distinguished between the two forms. Thus monoclonal antibody SA7 bound the 6.5 S form selectively, whereas SA57 recognized the 8 S form. The selective binding of the 8 S form to heparin suggests that, within the parasite, this form may be associated with the extracellular matrix of the musculature.
ESTHER : Tarrab-Hazdai_1999_Biochem.J_344 Pt 3_945
PubMedSearch : Tarrab-Hazdai_1999_Biochem.J_344 Pt 3_945
PubMedID: 10585885

Title : Targeted cross-linking of a molten globule form of acetylcholinesterase by the virucidal agent hypericin - Weiner_1999_Biochemistry_38_11401
Author(s) : Weiner L , Roth E , Mazur Y , Silman I
Ref : Biochemistry , 38 :11401 , 1999
Abstract : The natural product hypericin is a photosensitive polycyclic aromatic dione compound, which has been widely investigated because of its virucidal and antitumor properties. Although it has been suggested that singlet oxygen or a radical species might be responsible for its biological action, its mechanism of action remains unknown. Due to its amphiphilic characteristics, we considered the possibility that it might interact preferentially with partially unfolded proteins which exhibit exposed hydrophobic surfaces. We here demonstrate that hypericin binds to a molten globule species generated from Torpedo acetylcholinesterase, but not to the corresponding native enzyme. Irradiation with visible light, under aerobic conditions, causes chemical cross-linking of the catalytic subunits, to dimers and heavier species, under conditions where no cross-linking is observed for the native enzyme. Both anaerobiosis and sodium azide greatly reduce the extent of cross-linking, suggesting that singlet oxygen is responsible for the phenomenon. This agrees with our observation, using spin traps, that mainly singlet oxygen is produced by the complex of hypericin with the molten globule of acetylcholinesterase. Cross-linking is enhanced in the presence of liposomes to which the molten globule of acetylcholinesterase is quantitatively adsorbed. This may be due to high local concentrations of both hypericin and the protein resulting in close proximity, and hence in a high yield of cross-linking. Molten globule species are believed to be intermediates in both protein folding and translocation through biological membranes. Thus, hypericin may serve as a valuable tool for trapping such intermediates. This might also explain its therapeutic effectiveness toward virus-infected or tumor cells.
ESTHER : Weiner_1999_Biochemistry_38_11401
PubMedSearch : Weiner_1999_Biochemistry_38_11401
PubMedID: 10471290

Title : 3D Structure at 2.7 Resolution of Native and E202Q Mutant Human Acetylcholinesterase Complexed with Fasciculin-II -
Author(s) : Kryger G , Giles K , Harel M , Toker L , Velan B , Lazar A , Kronman C , Barak D , Ariel N , Shafferman A , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :323 , 1998
PubMedID:

Title : Liposome-catalyzed unfolding of acetylcholinesterase from Bungarus fasciatus - Shin_1998_Biochemistry_37_4310
Author(s) : Shin I , Silman I , Bon C , Weiner L
Ref : Biochemistry , 37 :4310 , 1998
Abstract : The kinetics of thermal inactivation of acetylcholinesterase from the venom of the snake, Bungarus fasciatus, were studied at 45-54 degrees C. An Arrhenius plot reveals an activation energy of 113 kcal/mol. The thermally denatured enzyme displays the spectroscopic characteristics of a partially unfolded 'molten globule' state. The rate of thermal denaturation is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, the energy barrier for the transition being lowered from 113 to 52 kcal/mol. In contrast to our findings for partially unfolded Torpedo californica acetylcholinesterase [Shin et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2848-2852], the thermally denatured snake enzyme does not remain bound to the liposomes but is released after unfolding and subsequently aggregates. The liposomes thus serve as catalysts for unfolding of the snake enzyme, and its rate of unfolding in the presence of liposomes can be described by the Michaelis-Menten equation (Km = 8 x 10(-7) M). The phospholipid vesicles display a catalytic turnover number of kcat approximately 4 min-1, assuming 15 binding sites per vesicle for the snake acetylcholinesterase.
ESTHER : Shin_1998_Biochemistry_37_4310
PubMedSearch : Shin_1998_Biochemistry_37_4310
PubMedID: 9556345

Title : Irwin B. Wilson Plenary Lecture Structural and Functional Studies on Acetylcholinesterase -
Author(s) : Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :25 , 1998
PubMedID:

Title : Electrotactins: a class of adhesion proteins with conserved electrostatic and structural motifs - Botti_1998_Protein.Eng_11_415
Author(s) : Botti SA , Felder CE , Sussman JL , Silman I
Ref : Protein Engineering , 11 :415 , 1998
Abstract : The concept of an electrostatic motif on the surface of biological macromolecules as a definite topographical pattern of electrostatic potentials in three-dimensional space, provides a powerful tool for identification of functionally important regions on the surface of structurally related macromolecules. Using this approach, we identify a functional region common to cholinesterases (ChEs) and to a set of neural cell-adhesion proteins that have been suggested to be structurally related to cholinesterases due to their high sequence similarity, but lacking the key catalytically active serine. Quantitative analysis of the electrostatic surface potential in the area surrounding the entrance to the active site of acetylcholinesterase, and in the analogous zone for the ChE-like domain of the adhesion proteins reveals very good correlation. These findings, examined in the context of previous evidence involving this same region in a possible cell-recognition function for ChEs, leads us to define a class of adhesion proteins which we have named 'electrotactins'.
ESTHER : Botti_1998_Protein.Eng_11_415
PubMedSearch : Botti_1998_Protein.Eng_11_415
PubMedID: 9725619

Title : 3D Structure of a Complex of Human Acetylcholinesterase with Fasciculin-II at 2.7 Resolution -
Author(s) : Kryger G , Giles K , Harel M , Toker L , Velan B , Lazar A , Kronman C , Barak D , Ariel N , Shafferman A , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :370 , 1998
PubMedID:

Title : Crystal Structures of Complexes of E2020-Related Compounds with Torpedo Californica Acetylcholinesterase -
Author(s) : Greenblatt HM , Kryger G , Harel M , Lewis T , Taylor J , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :371-371 , 1998
PubMedID:

Title : Alternative Crystal Forms of Torpedo Californica Acetylcholinesterase -
Author(s) : Raves ML , Greenblatt HM , Kryger G , Nicolas A , Ravelli RB , Harel M , Kroon J , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :372 , 1998
PubMedID:

Title : Kinetic and X-Ray Crystallographic Studies of the Binding of ENA-713 to Torpedo Californica Acetylcholinesterase -
Author(s) : Bar-On P , Harel M , Millard CB , Enz A , Sussman JL , Silman I
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. :373 , 1998
PubMedID:

Title : Kinetic and structural studies on the interaction of the anti-Alzheimer drug, ENA-713, with Torpedo californica acetylcholinesterase -
Author(s) : Bar-On P , Harel M , Millard CB , Enz A , Sussman JL , Silman I
Ref : Journal de Physiologie (Paris) , 92 :406 , 1998
PubMedID:

Title : Crystal Structures of Aged Phosphorylated and Phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :425 , 1998
PubMedID:

Title : Crystal structure of Aged phosphorylated and phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
Ref : In Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :425 , 1998
PubMedID:
Gene_locus related to this paper: torca-ACHE

Title : Surface Residues Near the Peripheral Site Affect the Stability of Torpedo Acetylcholinesterase -
Author(s) : Morel N , Bon S , Sussman JL , Massoulie J , Silman I
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. :435 , 1998
PubMedID:

Title : Membrane-Promoted Unfolding of Torpedo Californica and Bungarus Fasciatus Acetylcholinesterase -
Author(s) : Shin I , Silman I , Bon C , Weiner L
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. :438 , 1998
PubMedID:

Title : Static laue diffraction studies on acetylcholinesterase - Ravelli_1998_Acta.Crystallogr.D.Biol.Crystallogr_54_1359
Author(s) : Ravelli RB , Raves ML , Ren Z , Bourgeois D , Roth M , Kroon J , Silman I , Sussman JL
Ref : Acta Crystallographica D Biol Crystallogr , 54 :1359 , 1998
Abstract : Acetylcholinesterase (AChE) is one of nature's fastest enzymes, despite the fact that its three-dimensional structure reveals its active site to be deeply sequestered within the molecule. This raises questions with respect to traffic of substrate to, and products from, the active site, which may be investigated by time-resolved crystallography. In order to address one aspect of the feasibility of performing time-resolved studies on AChE, a data set has been collected using the Laue technique on a trigonal crystal of Torpedo californica AChE soaked with the reversible inhibitor edrophonium, using a total X-ray exposure time of 24 ms. Electron-density maps obtained from the Laue data, which are of surprisingly good quality compared with similar maps from monochromatic data, show essentially the same features. They clearly reveal the bound ligand, as well as a structural change in the conformation of the active-site Ser200 induced upon binding.
ESTHER : Ravelli_1998_Acta.Crystallogr.D.Biol.Crystallogr_54_1359
PubMedSearch : Ravelli_1998_Acta.Crystallogr.D.Biol.Crystallogr_54_1359
PubMedID: 10089512
Gene_locus related to this paper: torca-ACHE

Title : Acetylcholinesterase of S. mansoni (Trematode). Interaction of globular species with glycosaminoglycans -
Author(s) : Tarrab-Hazdai R , Silman I , Toker L , Schechtman D , Arnon R
Ref : Journal de Physiologie (Paris) , 92 :501 , 1998
PubMedID:

Title : Electrostatic homology modelling of a set of ChE-like neural adhesion proteins identifies a shared annular motif with ChEs. Structural implications for a cell-recognition role of ChEs -
Author(s) : Botti SA , Felder CE , Sussman JL , Silman I
Ref : Journal de Physiologie (Paris) , 92 :414 , 1998
PubMedID:

Title : Assembly of Acetylcholinesterase Subunits in vitro -
Author(s) : Giles K , Ben-Yohanan R , Velan B , Shafferman A , Sussman JL , Silman I
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. :442 , 1998
PubMedID:

Title : Quaternary Structure of Tetrameric Acetylcholinesterase -
Author(s) : Raves ML , Giles K , Schrag JD , Schmid MF , Phillips JN, Jr. , Chiu W , Howard AJ , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :351 , 1998
PubMedID:
Gene_locus related to this paper: eleel-ACHE

Title : The Conjunction of a Conserved Electrostatic Motif and a Common Cholinesterase Fold Defines a Class of Adhesion Proteins -
Author(s) : Botti SA , Felder CE , Sussman JL , Silman I
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. :448 , 1998
PubMedID:

Title : Expression and Tissue Distribution of Cholinesterases Via EST Analysis -
Author(s) : Giles K , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :450 , 1998
PubMedID:

Title : Crystal Structures of Aged Phosphorylated and Phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :454 , 1998
PubMedID:

Title : Three-dimensional structure of a complex of E2020 with acetylcholinesterase from Torpedo californica - Kryger_1998_J.Physiol.Paris_92_191
Author(s) : Kryger G , Silman I , Sussman JL
Ref : Journal de Physiologie Paris , 92 :191 , 1998
Abstract : The 3D structure of a complex of the anti-Alzheimer drug, E2020, also known as Aricept, with Torpedo californica acetylcholinesterase is reported. The X-ray structure, at 2.5 A resolution, shows that the elongated E2020 molecule spans the entire length of the active-site gorge of the enzyme. It thus interacts with both the 'anionic' subsite, at the bottom of the gorge, and with the peripheral anionic site, near its entrance, via aromatic stacking interactions with conserved aromatic residues. It does not interact directly with either the catalytic triad or with the 'oxyanion hole'. Although E2020 is a chiral molecule, and both the S and R enantiomers have similar affinity for the enzyme, only the R enantiomer is bound within the active-site gorge when the racemate is soaked into the crystal. The selectivity of E2020 for acetylcholinesterase, relative to butyrylcholinesterase, can be ascribed primarily to its interactions with Trp279 and Phe330, which are absent in the latter.
ESTHER : Kryger_1998_J.Physiol.Paris_92_191
PubMedSearch : Kryger_1998_J.Physiol.Paris_92_191
PubMedID: 9789806

Title : 3D Structure of a Complex of the Anti-Alzheimer Drug, E2020, with Acetylcholinesterase at 2.5 Resolution -
Author(s) : Kryger G , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :469 , 1998
PubMedID:

Title : An Integrated Model for the Molecular Traffic through the Active Site of Cholinesterases -
Author(s) : Botti SA , Felder CE , Lifson S , Sussman JL , Silman I
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. :227 , 1998
PubMedID:

Title : Activity of Torpedo Californica Acetylcholinesterase in the Crystalline State -
Author(s) : Nicolas A , Millard CB , Raves ML , Ravelli RB , Kroon J , Silman I , Sussman JL
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :230 , 1998
PubMedID:

Title : Membrane-promoted unfolding of acetylcholinesterase: a possible mechanism for insertion into the lipid bilayer - Shin_1997_Proc.Natl.Acad.Sci.U.S.A_94_2848
Author(s) : Shin I , Kreimer DI , Silman I , Weiner L
Ref : Proceedings of the National Academy of Sciences of the United States of America , 94 :2848 , 1997
Abstract : Acetylcholinesterase from Torpedo californica partially unfolds to a state with the physicochemical characteristics of a "molten globule" upon mild thermal denaturation or upon chemical modification of a single non-conserved buried cysteine residue, Cys231. The protein in this state binds tightly to liposomes. It is here shown that the rate of unfolding is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, with concomitant incorporation of the protein into the lipid bilayer. Arrhenius plots reveal that in the presence of the liposomes the energy barrier for transition from the native to the molten globule state is lowered from 145 to 47 kcal/mol. Chemical modification of Cys231 by mercuric chloride produces initially a quasinative state of Torpedo acetylcholinesterase which, at room temperature, undergoes spontaneous transition to a molten globule state with a half-life of 1-2 hr. This permitted temporal resolution of interaction of the quasi-native state with the membrane from the transition of the membrane-bound protein to the molten globule state. The data presented here suggest that either the native enzyme, or a quasi-native state with which it is in equilibrium, interacts with the liposome, which then promotes a fast transition to the membrane-bound molten globule state by lowering the energy barrier for the transition. These findings raise the possibility that the membrane itself, by lowering the energy barrier for transition to a partially unfolded state, may play an active posttranslational role in insertion and translocation of proteins in situ.
ESTHER : Shin_1997_Proc.Natl.Acad.Sci.U.S.A_94_2848
PubMedSearch : Shin_1997_Proc.Natl.Acad.Sci.U.S.A_94_2848
PubMedID: 9096309

Title : Structure of acetylcholinesterase complexed with the nootropic alkaloid, (-)-huperzine A - Raves_1997_Nat.Struct.Biol_4_57
Author(s) : Raves ML , Harel M , Pang YP , Silman I , Kozikowski AP , Sussman JL
Ref : Nat Struct Biol , 4 :57 , 1997
Abstract : (-)-Huperzine A (HupA) is found in an extract from a club moss that has been used for centuries in Chinese folk medicine. Its action has been attributed to its ability to strongly inhibit acetylcholinesterase (AChE). The crystal structure of the complex of AChE with optically pure HupA at 2.5 A resolution shows an unexpected orientation for the inhibitor with surprisingly few strong direct interactions with protein residues to explain its high affinity. This structure is compared to the native structure of AChE devoid of any inhibitor as determined to the same resolution. An analysis of the affinities of structural analogues of HupA, correlated with their interactions with the protein, shows the importance of individual hydrophobic interactions between HupA and aromatic residues in the active-site gorge of AChE.
ESTHER : Raves_1997_Nat.Struct.Biol_4_57
PubMedSearch : Raves_1997_Nat.Struct.Biol_4_57
PubMedID: 8989325
Gene_locus related to this paper: torca-ACHE

Title : External and internal electrostatic potentials of cholinesterase models - Felder_1997_J.Mol.Graph.Model_15_318
Author(s) : Felder CE , Botti SA , Lifson S , Silman I , Sussman JL
Ref : J Mol Graph Model , 15 :318 , 1997
Abstract : The electrostatic potentials for the three-dimensional structures of cholinesterases from various species were calculated, using the Delphi algorithm, on the basis of the Poisson-Boltzmann equation. We used structures for Torpedo californica and mouse acetylcholinesterase, and built homology models of the human, Bungarus fasciatus, and Drosophila melanogaster acetylcholinesterases and human butyrylcholinesterase. All these structures reveal a negative external surface potential, in the area around the entrance to the active-site gorge, that becomes more negative as the rim of the gorge is approached. Moreover, in all cases, the potential becomes increasingly more negative along the central axis running down the gorge, and is largest at the base of the gorge, near the active site. Ten key acidic residues conserved in the sequence alignments of AChE from various species, both in the surface area near the entrance of the active-site gorge and at its base, appear to be primarily responsible for these potentials. The potentials are highly correlated among the structures examined, down to sequence identities as low as 35%. This indicates that they are a conserved property of the cholinesterase family, could serve to attract the positively charged substrate into and down the gorge to the active site, and may play other roles important for cholinesterase function.
ESTHER : Felder_1997_J.Mol.Graph.Model_15_318
PubMedSearch : Felder_1997_J.Mol.Graph.Model_15_318
PubMedID: 9640563

Title : The X-ray structure of a transition state analog complex reveals the molecular origins of the catalytic power and substrate specificity of acetylcholinesterase. -
Author(s) : Harel M , Quinn DM , Nair HK , Silman I , Sussman JL
Ref : Journal of the American Chemical Society , 118 :2340 , 1996
PubMedID:
Gene_locus related to this paper: torca-ACHE

Title : Two partially unfolded states of Torpedo californica acetylcholinesterase - Kreimer_1996_Protein.Sci_5_1852
Author(s) : Kreimer DI , Shin I , Shnyrov VL , Villar E , Silman I , Weiner L
Ref : Protein Science , 5 :1852 , 1996
Abstract : Chemical modification with sulfhydryl reagents of the single, nonconserved cysteine residue Cys231 in each subunit of a disulfide-linked dimer of Torpedo californica acetylcholinesterase produces a partially unfolded inactive state. Another partially unfolded state can be obtained by exposure of the enzyme to 1-2 M guanidine hydrochloride. Both these states display several important features of a molten globule, but differ in their spectroscopic (CD, intrinsic fluorescence) and hydrodynamic (Stokes radii) characteristics. With reversal of chemical modification of the former state or removal of denaturant from the latter, both states retain their physiochemical characteristics. Thus, acetylcholinesterase can exist in two molten globule states, both of which are long-lived under physiologic conditions without aggregating, and without either intraconverting or reverting to the native state. Both states undergo spontaneous intramolecular thioldisulfide exchange, implying that they are flexible. As revealed by differential scanning calorimetry, the state produced by chemical modification lacks any heat capacity peak, presumably due to aggregation during scanning, whereas the state produced by guanidine hydrochloride unfolds as a single cooperative unit, thermal transition being completely reversible. Sucrose gradient centrifugation reveals that reduction of the interchain disulfide of the native acetylcholinesterase dimer converts it to monomers, whereas, after such reduction, the two subunits remain completely associated in the partially unfolded state generated by guanidine hydrochloride, and partially associated in that produced by chemical modification. It is suggested that a novel hydrophobic core, generated across the subunit interfaces, is responsible for this noncovalent association. Transition from the unfolded state generated by chemical modification to that produced by guanidine hydrochloride is observed only in the presence of the denaturant, yielding, on extrapolation to zero guanidine hydrochloride, a high free energy barrier (ca. 23.8 kcal/mol) separating these two flexible, partially unfolded states.
ESTHER : Kreimer_1996_Protein.Sci_5_1852
PubMedSearch : Kreimer_1996_Protein.Sci_5_1852
PubMedID: 8880909

Title : Site-directed mutants designed to test back-door hypotheses of acetylcholinesterase function - Faerman_1996_FEBS.Lett_386_65
Author(s) : Faerman C , Ripoll D , Bon S , Le Feuvre Y , Morel N , Massoulie J , Sussman JL , Silman I
Ref : FEBS Letters , 386 :65 , 1996
Abstract : The location of the active site of the rapid enzyme, acetylcholinesterase, near the bottom of a deep and narrow gorge indicates that alternative routes may exist for traffic of substrate, products or solute into and out of the gorge. Molecular dynamics suggest the existence of a shutter-like back door near Trp84, a key- residue in the binding site for acetylcholine, in the Torpedo californica enzyme. The homology of the omega loop, bearing Trp84, with the lid which sequesters the substrate in neutral lipases displaying structural homology with acetylcholinesterase, suggests a flap-like back door. Both possibilities were examined by site-directed mutagenesis. The shutter-like back door was tested by generating a salt bridge which might impede opening of the shutter. The flap-like back door was tested by de novo insertion of a disulfide bridge which tethered the omega loop to the body of the enzyme. Neither type of mutation produced significant changes in catalytic activity, thus failing to provide experimental support for either back door model. Molecular dynamics revealed, however, substantial mobility of the omega loop in the immediate vicinity of Trp84, even when the loop was tethered, supporting the possibility that access to the active site, involving limited movement of a segment of the loop, is indeed possible.
ESTHER : Faerman_1996_FEBS.Lett_386_65
PubMedSearch : Faerman_1996_FEBS.Lett_386_65
PubMedID: 8635606

Title : Biochemical evaluation of photolabile precursors of choline and of carbamylcholine for potential time-resolved crystallographic studies on cholinesterases - Peng_1996_Biochemistry_35_10854
Author(s) : Peng L , Silman I , Sussman JL , Goeldner M
Ref : Biochemistry , 35 :10854 , 1996
Abstract : Acetylcholinesterase and butyrylcholinesterase both rapidly hydrolyze the neurotransmitter acetylcholine. The unusual three-dimensional structure of acetylcholinesterase, in which the active site is located at the bottom of a deep and narrow gorge, raises cogent questions concerning traffic of the substrate, acetylcholine, and the products, choline and acetate, to and from the active site. Time-resolved crystallography offers a promising experimental approach to investigate this issue but requires a suitable triggering mechanism to ensure efficient and synchronized initiation of the dynamic process being monitored. Here we characterize the properties of two photolabile triggers which may serve as tools in time-resolved crystallographic studies of the cholinesterases. These compounds are 2-nitrobenzyl derivatives of choline and of carbamylcholine, which generate choline and carbamylcholine, respectively, upon photochemical fragmentation. Both photolabile compounds are reversible inhibitors, which bind at the active sites of acetylcholinesterase and butyrylcholinesterase with inhibition constants in the micromolar range, and both photofragmentation processes occur rapidly and with a high quantum yield, without substantial photochemical damage to the enzymes. Photolysis both of acetylcholinesterase and of butyrylcholinesterase, complexed with a 2-nitrobenzyl derivative of choline, resulted in regeneration of enzymic activity. Photolysis of acetylcholinesterase complexed with the 2-nitrobenzyl derivative of carbamylcholine led to time-dependent inactivation, resulting from carbamylation of acetylcholinesterase, which could be reversed upon dilution, due to decarbamylation. Both sets of experiments demonstrated release of choline within the active site. In the former case, choline was produced photochemically at the active site. In the latter case, choline was generated enzymatically, within the active site, concomitantly with carbamylation of the acetylcholinesterase. The two photolabile compounds may thus serve as complementary probes for time-resolved studies of the route of product release from the active sites of the cholinesterases.
ESTHER : Peng_1996_Biochemistry_35_10854
PubMedSearch : Peng_1996_Biochemistry_35_10854
PubMedID: 8718877

Title : Interaction of partially unfolded forms of Torpedo acetylcholinesterase with liposomes - Shin_1996_Prot.Sci_5_42
Author(s) : Shin I , Silman I , Weiner L
Ref : Protein Science , 5 :42 , 1996
Abstract : A water-soluble dimeric form of acetylcholinesterase from electric organ tissue of Torpedo californica was obtained by solubilization with phosphatidylinositol-specific phospholipase C of the glycophosphatidylinositol-anchored species, followed by purification by affinity chromatography. The water-soluble species, in its catalytically active native conformation, did not interact with unilamellar vesicles of dimyristoylphosphatidylcholine. We previously showed that either chemical modification or exposure to low concentrations of guanidine hydrochloride converted the native enzyme to compact, partially unfolded species with the physicochemical characteristics of the molten globule state. In the present study, it was shown that such molten globule species, whether produced by mild denaturation or by chemical modification, interacted efficiently with small unilamellar vesicles. Binding was not accompanied by significant vesicle fusion, but transient leakage occurred at the time of binding. The bound acetylcholinesterase reduced the transition temperature of the vesicles slightly, and NMR data suggested that it interacted primarily with the head-group region of the bilayer. The effects of tryptic digestion of the bound acetycholinesterase were monitored by gel electrophoresis under denaturing conditions. It was found that a single polypeptide, of mass approximately 5 kDa, remained associated with the vesicles. Sequencing revealed that this is a tryptic peptide corresponding to the sequence Glu 268-Lys 315. This polypeptide contains the longest hydrophobic sequence in the protein, Leu 274-Met 308, as identified on the basis of hydropathy plots. Inspection of the three-dimensional structure of acetylcholinesterase reveals that this hydrophobic sequence is largely devoid of tertiary structure and is localized primarily on the surface of the protein. It is suggested that this hydrophobic sequence is aligned parallel to the surface of the vesicle membrane, with nonpolar residues undergoing shallow penetration into the bilayer.
ESTHER : Shin_1996_Prot.Sci_5_42
PubMedSearch : Shin_1996_Prot.Sci_5_42
PubMedID: 8771195

Title : Electrooptical measurements demonstrate a large permanent dipole moment associated with acetylcholinesterase - Porschke_1996_Biophys.J_70_1603
Author(s) : Porschke D , Creminon C , Cousin X , Bon C , Sussman JL , Silman I
Ref : Biophysical Journal , 70 :1603 , 1996
Abstract : Acetylcholinesterase (AChE) from krait (Bungarus fasciatus) venom is a soluble, nonamphiphilic monomer of 72 kDa. This snake venom AChE has been analyzed by measurements of the stationary and the transient electric dichroism at different field strengths. The stationary values of the dichroism are consistent with the orientation function for permanent dipoles and are not consistent with the orientation function for induced dipoles. The permanent dipole moment obtained by least-squares fits for a buffer containing 5 mM MES is 1000 D, after correction for the internal directing field, assuming a spherical shape of the protein. The dipole moment decreases with increasing buffer concentration to 880 D at 10 mM MES and 770 D at 20 mM MES. The dichroism decay time constant is 90 ns (+/- 10%) which is clearly larger than the value expected from the size/shape of the protein and indicates contributions from sugar residues attached to the protein. The dichroism rise times observed at low field strengths are larger than the decay times and, thus, support the assignment of a permanent dipole moment, although it has not been possible to approach the limit where the energy of the dipole in the electric field is sufficiently low compared to kT. The experimental value of the permanent dipole moment is similar to that calculated for a model structure of Bungarus fasciatus AChE, which has been constructed from its amino and acid sequence, in analogy to the crystal structure of AChE from Torpedo californica.
ESTHER : Porschke_1996_Biophys.J_70_1603
PubMedSearch : Porschke_1996_Biophys.J_70_1603
PubMedID: 8785319

Title : Structures of Complexes of Acetylcholinesterase with Covalently and Non-Covalently Bound Inhibitors -
Author(s) : Sussman JL , Harel M , Raves ML , Quinn DM , Nair HK , Silman I
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. :59 , 1995
PubMedID:

Title : Electrostatic Properties of Human Acetylcholinesterase -
Author(s) : Ripoll DR , Faerman CH , Gillilan R , Silman I , Sussman JL
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. :67 , 1995
PubMedID:

Title : Studies on Partially Unfolded States of Torpedo californica Acetylcholinesterase -
Author(s) : Silman I , Kreimer DI , Shin I , Dolginova EA , Roth E , Goldfarb D , Szosenfogel R , Raves ML , Sussman JL , Borochov N , Weiner L
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. :77 , 1995
PubMedID:

Title : Irreversible thermal denaturation of Torpedo californica acetylcholinesterase - Kreimer_1995_Prot.Sci_4_2349
Author(s) : Kreimer DI , Shnyrov VL , Villar E , Silman I , Weiner L
Ref : Protein Science , 4 :2349 , 1995
Abstract : Thermal denaturation of Torpedo californica acetylcholinesterase, a disulfide-linked homodimer with 537 amino acids in each subunit, was studied by differential scanning calorimetry. It displays a single calorimetric peak that is completely irreversible, the shape and temperature maximum depending on the scan rate. Thus, thermal denaturation of acetylcholinesterase is an irreversible process, under kinetic control, which is described well by the two-state kinetic scheme N-->D, with activation energy 131 +/- 8 kcal/mol. Analysis of the kinetics of denaturation in the thermal transition temperature range, by monitoring loss of enzymic activity, yields activation energy of 121 +/- 20 kcal/mol, similar to the value obtained by differential scanning calorimetry. Thermally denatured acetylcholinesterase displays spectroscopic characteristics typical of a molten globule state, similar to those of partially unfolded enzyme obtained by modification with thiol-specific reagents. Evidence is presented that the partially unfolded states produced by the two different treatments are thermodynamically favored relative to the native state.
ESTHER : Kreimer_1995_Prot.Sci_4_2349
PubMedSearch : Kreimer_1995_Prot.Sci_4_2349
PubMedID: 8563632

Title : Crystal structure of an acetylcholinesterase-fasciculin complex: interaction of a three-fingered toxin from snake venom with its target - Harel_1995_Structure_3_1355
Author(s) : Harel M , Kleywegt GJ , Ravelli RB , Silman I , Sussman JL
Ref : Structure , 3 :1355 , 1995
Abstract : BACKGROUND Fasciculin (FAS), a 61-residue polypeptide purified from mamba venom, is a three-fingered toxin which is a powerful reversible inhibitor of acetylcholinesterase (AChE). Solution of the three-dimensional structure of the AChE/FAS complex would provide the first structure of a three-fingered toxin complexed with its target. RESULTS: The structure of a complex between Torpedo californica AChE and fasciculin-II (FAS-II), from the venom of the green mamba (Dendroaspis angusticeps) was solved by molecular replacement techniques, and refined at 3.0 A resolution to an R-factor of 0.231. The structure reveals a stoichiometric complex with one FAS molecule bound to each AChE subunit. The AChE and FAS conformations in the complex are very similar to those in their isolated structures. FAS is bound at the 'peripheral' anionic site of AChE, sealing the narrow gorge leading to the active site, with the dipole moments of the two molecules roughly aligned. The high affinity of FAS for AChE is due to a remarkable surface complementarity, involving a large contact area (approximately 2000 A2) and many residues either unique to FAS or rare in other three-fingered toxins. The first loop, or finger, of FAS reaches down the outer surface of the thin aspect of the gorge. The second loop inserts into the gorge, with an unusual stacking interaction between Met33 in FAS and Trp279 in AChE. The third loop points away from the gorge, but the C-terminal residue makes contact with the enzyme. CONCLUSIONS: Two conserved aromatic residues in the AChE peripheral anionic site make important contacts with FAS. The absence of these residues from chicken and insect AChEs and from butyrylcholinesterase explains the very large reduction in the affinity of these enzymes for FAS. Several basic residues in FAS make important contacts with AChE. The complementarity between FAS and AChE is unusual, inasmuch as it involves a number of charged residues, but lacks any intermolecular salt linkages.
ESTHER : Harel_1995_Structure_3_1355
PubMedSearch : Harel_1995_Structure_3_1355
PubMedID: 8747462
Gene_locus related to this paper: torca-ACHE

Title : The activity of an endoplasmic reticulum-localized pool of acetylcholinesterase is modulated by heat shock - Eichler_1995_J.Biol.Chem_270_4466
Author(s) : Eichler J , Silman I
Ref : Journal of Biological Chemistry , 270 :4466 , 1995
Abstract : Primary cultures prepared from embryonic chick pectoral muscle were subjected to heat shock, and the effect on acetylcholinesterase activity in the cultures was examined. A rapid recovery in enzyme activity was observed soon after an initial heat shock-induced drop and was shown to be independent of de novo synthesis of protein, since it could occur in the presence of an inhibitor of protein synthesis. Lectin binding and sucrose gradient centrifugation studies suggested that molecular monomers and dimers found in the endoplasmic reticulum are involved in the observed recovery of acetylcholinesterase activity. Enhanced activation of a pre-existing pool of inactive enzyme was clearly not the main agent of the recovery in enzymic activity. Recovery relied principally on restoration of the activity of previously active, heat-denatured acetylcholinesterase molecules found in the endoplasmic reticulum. Possible agents involved in the recovery of enzymatic activity might be heat shock proteins acting as molecular chaperones.
ESTHER : Eichler_1995_J.Biol.Chem_270_4466
PubMedSearch : Eichler_1995_J.Biol.Chem_270_4466
PubMedID: 7876213

Title : Differential effects of peripheral site ligands on Torpedo and chicken acetylcholinesterase - Eichler_1994_Mol.Pharmacol_45_335
Author(s) : Eichler J , Anselmet A , Sussman JL , Massoulie J , Silman I
Ref : Molecular Pharmacology , 45 :335 , 1994
Abstract : Comparison of the effect of three 'peripheral' site ligands, propidium, d-tubocurarine, and gallamine, on acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) of Torpedo and chicken shows that all three are substantially more effective inhibitors of the Torpedo enzyme than of the chicken enzyme. In contrast, edrophonium, which is directed to the "anionic" subsite of the active site, inhibits the chicken and Torpedo enzymes equally effectively. Two bisquaternary ligands, decamethonium and 1,5-bis(4-allydimethylammoniumphenyl)pentan-3-one dibromide, which are believed to bridge the anionic subsite of the active site and the "peripheral" anionic site, are much weaker inhibitors of the chicken enzyme than of Torpedo acetylcholinesterase, whereas the shorter bisquaternary ligand hexamethonium inhibits the two enzymes similarly. The concentration dependence of activity towards the natural substrate acetylcholine is almost identical for the two enzymes, whereas substrate inhibition of chicken acetylcholinesterase is somewhat weaker than that of the Torpedo enzyme. The experimental data can be rationalized on the basis of the three-dimensional structure of the Torpedo enzyme and alignment of the chicken and Torpedo sequences; it is suggested that the absence, in the chicken enzyme, of two aromatic residues, Tyr-70 and Trp-279, that contribute to the peripheral site of Torpedo acetylcholinesterase is responsible for the differential effects of peripheral site ligands on the two enzymes.
ESTHER : Eichler_1994_Mol.Pharmacol_45_335
PubMedSearch : Eichler_1994_Mol.Pharmacol_45_335
PubMedID: 8114681

Title : Three-dimensional structures of acetylcholinesterase and of its complexes with anticholinesterase agents -
Author(s) : Silman I , Harel M , Axelsen PH , Raves ML , Sussman JL
Ref : Biochemical Society Transactions , 22 :745 , 1994
PubMedID: 7821677

Title : Oxidative stress transforms acetylcholinesterase to a molten-globule-like state - Weiner_1994_Biochem.Biophys.Res.Commun_198_915
Author(s) : Weiner L , Kreimer D , Roth E , Silman I
Ref : Biochemical & Biophysical Research Communications , 198 :915 , 1994
Abstract : Exposure of purified acetylcholinesterase from Torpedo california to a system generating oxygen radicals (viz. ascorbic acid/Fe(EDTA)2/H2O2) inactivated the enzyme. The enzyme retained its native dimeric form, but electrophoresis under denaturing conditions showed some cleavage of peptide bonds. Spectroscopic characterization revealed a shift to the red in the intrinsic fluorescence emission peak, a large decrease in molar ellipticity in the near UV with a much smaller decrease in the far UV, and increased binding of the amphiphilic probe, 1-anilino-8-naphthalene sulfonate, all relative to native enzyme. The treated enzyme was also highly susceptible to proteolysis. These data show that oxygen radical treatment converts acetylcholinesterase to a partially unfolded state, which retains most of its secondary structure but lacks substantial tertiary structure, thus resembling a 'molten globule' state. This model system may offer a mechanism for explaining the consequences of oxidative stress in vivo: partially unfolded proteins generated by oxidative stress may interact with molecular chaperons of the heat shock family, thus activating the heat-shock factor and, thereby, activating heat-shock genes.
ESTHER : Weiner_1994_Biochem.Biophys.Res.Commun_198_915
PubMedSearch : Weiner_1994_Biochem.Biophys.Res.Commun_198_915
PubMedID: 8117296

Title : Novel GPI structures of the membrane anchor of acetylcholinesterase from the electric organ of Torpedo californica - Mehlert_1994_Brazilian.J.Med.Biol.Res_27_243
Author(s) : Mehlert A , Varon L , Silman I , Homans SW , Ferguson MA
Ref : Brazilian Journal of Medical & Biological Research , 27 :243 , 1994
Abstract : The structure of the glycan moiety of the glycosylphosphatidylinositol (GPI) membrane anchor from Torpedo californica electric organ acetylcholinesterase was solved using nuclear magnetic resonance (NMR), methylation analysis, and chemical and enzymic microsequencing. Two structures were found to be present: Glc alpha 1-2 Man alpha 1-2 Man alpha 1-6 Man alpha 1-4 GlcN alpha 1-6myo-inositol, and Glc alpha 1-2 Man alpha 1-2 Man alpha 1-6 (GalNAc beta 1-4) Man alpha 1-4 GlcN alpha 1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule.
ESTHER : Mehlert_1994_Brazilian.J.Med.Biol.Res_27_243
PubMedSearch : Mehlert_1994_Brazilian.J.Med.Biol.Res_27_243
PubMedID: 8081237

Title : A metastable state of Torpedo californica acetylcholinesterase generated by modification with organomercurials - Kreimer_1994_Biochemistry_33_14407
Author(s) : Kreimer DI , Dolginova EA , Raves ML , Sussman JL , Silman I , Weiner L
Ref : Biochemistry , 33 :14407 , 1994
Abstract : Chemical modification of Torpedo californica acetylcholinesterase by various sulfhydryl reagents results in its conversion to one of two principal states. One of these states, viz., that produced by disulfides and alkylating agents, is stable. The second state, produced by mercury derivatives, is metastable. At room temperature, it converts spontaneously, with a half-life of ca. 1 h, to a stable state similar to that produced by the disulfides and alkylating agents. Demodification of acetylcholinesterase freshly modified by mercurials, by its exposure to reduced glutathione, causes rapid release of the bound mercurial, with concomitant recovery of most of the enzymic activity of the native enzyme. In contrast, similar demodification of acetylcholinesterase modified by disulfides yields no detectable recovery of enzymic activity. Spectroscopic measurements, employing CD, intrinsic fluorescence, and binding of 1-anilino-8-naphthalenesulfonate, show that the state produced initially by mercurials is "native-like", whereas that produced by disulfides and alkylating agents, and after prolonged incubation of the mercurial-modified enzyme, is partially unfolded and displays many of the features of the "molten globule" state. Arrhenius plots show that the quasi-native state produced by organomercurials is separated by a low (5 kcal/mol) energy barrier from the native state, whereas the partially unfolded state is separated from the quasi-native state by a high energy barrier (ca. 50 kcal/mol). Comparison of the 3D structures of native acetylcholinesterase and of a heavy-atom derivative obtained with HgAc2 suggests that the mercurial-modified enzyme may be stabilized by additional interactions of the mercury atom attached to the free thiol group of Cys231, specifically with Ser228O gamma with the main-chain nitrogen and carbonyl oxygen of the same serine residue.
ESTHER : Kreimer_1994_Biochemistry_33_14407
PubMedSearch : Kreimer_1994_Biochemistry_33_14407
PubMedID: 7981200
Gene_locus related to this paper: torca-ACHE

Title : Structure and dynamics of the active site gorge of acetylcholinesterase: synergistic use of molecular dynamics simulation and X-ray crystallography - Axelsen_1994_Prot.Sci_3_188
Author(s) : Axelsen PH , Harel M , Silman I , Sussman JL
Ref : Protein Science , 3 :188 , 1994
Abstract : The active site of acetylcholinesterase (AChE) from Torpedo californica is located 20 A from the enzyme surface at the bottom of a narrow gorge. To understand the role of this gorge in the function of AChE, we have studied simulations of its molecular dynamics. When simulations were conducted with pure water filling the gorge, residues in the vicinity of the active site deviated quickly and markedly from the crystal structure. Further study of the original crystallographic data suggests that a bis-quaternary decamethonium (DECA) ion, acquired during enzyme purification, residues in the gorge. There is additional electron density within the gorge that may represent small bound cations. When DECA and 2 cations are placed within the gorge, the simulation and the crystal structure are dramatically reconciled. The small cations, more so than DECA, appear to stabilize part of the gorge wall through electrostatic interactions. This part of the gorge wall is relatively thin and may regulate substrate, product, and water movement through the active site.
ESTHER : Axelsen_1994_Prot.Sci_3_188
PubMedSearch : Axelsen_1994_Prot.Sci_3_188
PubMedID: 8003956

Title : A molten globule of Torpedo acetylcholinesterase undergoes thiol-disulfide exchange - Eichler_1994_J.Biol.Chem_269_30093
Author(s) : Eichler J , Kreimer DI , Varon L , Silman I , Weiner L
Ref : Journal of Biological Chemistry , 269 :30093 , 1994
Abstract : Torpedo acetylcholinesterase is a disulfide-linked homodimer containing three intramolecular disulfide bonds, as well as a single free thiol on Cys-231. We report that in a "molten globule" state, produced by 1.5 M guanidine hydrochloride, this enzyme undergoes rapid intramolecular thiol-disulfide exchange, in the absence of reducing agents, resulting in the production of novel species. Most strikingly, this results in appearance of enzyme monomers. Chemical modification of the free thiol group prevents these changes. Unfolded acetylcholinesterase, namely in 5 M guanidine hydrochloride, also undergoes intramolecular thiol-disulfide exchange, including production of enzyme monomers, but at a much lower rate. Our data show that the molten globule state, in contrast to the native and unfolded states, is both compact and flexible, thus being especially amenable to thiol-disulfide exchange.
ESTHER : Eichler_1994_J.Biol.Chem_269_30093
PubMedSearch : Eichler_1994_J.Biol.Chem_269_30093
PubMedID: 7982909

Title : Two-state transition between molten globule and unfolded states of acetylcholinesterase as monitored by electron paramagnetic resonance spectroscopy - Kreimer_1994_Proc.Natl.Acad.Sci.U.S.A_91_12145
Author(s) : Kreimer DI , Szosenfogel R , Goldfarb D , Silman I , Weiner L
Ref : Proceedings of the National Academy of Sciences of the United States of America , 91 :12145 , 1994
Abstract : Cys-231 of Torpedo californica acetylcholinesterase (EC 3.1.1.7) was selectively labeled with the mercury derivative of a stable nitroxyl radical. In 1.5 M guanidinium chloride, this conjugate exists in a molten globule state (MG), whereas in 5 M denaturant, it is in an unfolded state (U). The transition between the two states is reversible. In the MG, the label is highly immobilized, whereas in the U, it is almost freely rotating. The clearly distinct electron paramagnetic resonance (EPR) spectra of the two states permits the study of this transition. Upon elevating the guanidinium chloride concentration, a decrease in the EPR signal of the MG occurs concomitantly with an increase in the U signal, the total intensity of the EPR spectra remaining constant. This behavior is characteristic of a two-state transition. The thermodynamic characteristics of this transition (delta G0 and m), whether estimated directly from the EPR data or from both CD and fluorescence data analyzed by assuming a two-state scheme, are in good agreement.
ESTHER : Kreimer_1994_Proc.Natl.Acad.Sci.U.S.A_91_12145
PubMedSearch : Kreimer_1994_Proc.Natl.Acad.Sci.U.S.A_91_12145
PubMedID: 7991597

Title : Open back door in a molecular dynamics simulation of acetylcholinesterase - Gilson_1994_Science_263_1276
Author(s) : Gilson MK , Straatsma TP , McCammon JA , Ripoll DR , Faerman CH , Axelsen PH , Silman I , Sussman JL
Ref : Science , 263 :1276 , 1994
Abstract : The enzyme acetylcholinesterase generates a strong electrostatic field that can attract the cationic substrate acetylcholine to the active site. However, the long and narrow active site gorge seems inconsistent with the enzyme's high catalytic rate. A molecular dynamics simulation of acetylcholinesterase in water reveals the transient opening of a short channel, large enough to pass a water molecule, through a thin wall of the active site near tryptophan-84. This simulation suggests that substrate, products, or solvent could move through this "back door," in addition to the entrance revealed by the crystallographic structure. Electrostatic calculations show a strong field at the back door, oriented to attract the substrate and the reaction product choline and to repel the other reaction product, acetate. Analysis of the open back door conformation suggests a mutation that could seal the back door and thus test the hypothesis that thermal motion of this enzyme may open multiple routes of access to its active site.
ESTHER : Gilson_1994_Science_263_1276
PubMedSearch : Gilson_1994_Science_263_1276
PubMedID: 8122110

Title : An electrostatic mechanism for substrate guidance down the aromatic gorge of acetylcholinesterase - Ripoll_1993_Proc.Natl.Acad.Sci.U.S.A_90_5128
Author(s) : Ripoll DR , Faerman CH , Axelsen PH , Silman I , Sussman JL
Ref : Proceedings of the National Academy of Sciences of the United States of America , 90 :5128 , 1993
Abstract : Electrostatic calculations based on the recently solved crystal structure of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) indicate that this enzyme has a strong electrostatic dipole. The dipole is aligned with the gorge leading to its active site, so that a positively charged substrate will be drawn to the active site by its electrostatic field. Within the gorge, aromatic side chains appear to shield the substrate from direct interaction with most of the negatively charged residues that give rise to the dipole. The affinity of quaternary ammonium compounds for aromatic rings, coupled with this electrostatic force, may work in concert to create a selective and efficient substrate-binding site in acetylcholinesterase and explain why the active site is situated at the bottom of a deep gorge lined with aromatic residues.
ESTHER : Ripoll_1993_Proc.Natl.Acad.Sci.U.S.A_90_5128
PubMedSearch : Ripoll_1993_Proc.Natl.Acad.Sci.U.S.A_90_5128
PubMedID: 8506359

Title : The structure of the glycosylphosphatidylinositol anchor from Torpedo californica acetylcholinesterase -
Author(s) : Mehlert A , Silman I , Homans SW , Ferguson MA
Ref : Biochemical Society Transactions , 21 , 1993
PubMedID: 8449327

Title : Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins - Cygler_1993_Protein.Sci_2_366
Author(s) : Cygler M , Schrag JD , Sussman JL , Harel M , Silman I , Gentry MK , Doctor BP
Ref : Protein Science , 2 :366 , 1993
Abstract : Based on the recently determined X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three-dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X-ray structures.
ESTHER : Cygler_1993_Protein.Sci_2_366
PubMedSearch : Cygler_1993_Protein.Sci_2_366
PubMedID: 8453375

Title : Structure of the glycosyl-phosphatidylinositol membrane anchor of acetylcholinesterase from the electric organ of the electric-fish, Torpedo californica - Mehlert_1993_Biochem.J_296_473
Author(s) : Mehlert A , Varon L , Silman I , Homans SW , Ferguson MA
Ref : Biochemical Journal , 296 :473 , 1993
Abstract : The structure of the glycan moiety of the glycosyl-phosphatidylinositol (GPI) membrane anchor from Torpedo californica (electric fish) electric-organ acetylcholinesterase was solved using n.m.r., methylation analysis and chemical and enzymic micro-sequencing. Two structures were found to be present: Glc alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol and Glc alpha 1-2Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN alpha 1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule.
ESTHER : Mehlert_1993_Biochem.J_296_473
PubMedSearch : Mehlert_1993_Biochem.J_296_473
PubMedID: 8257440

Title : Quaternary ligand binding to aromatic residues in the active-site gorge of acetylcholinesterase - Harel_1993_Proc.Natl.Acad.Sci.U.S.A_90_9031
Author(s) : Harel M , Schalk I , Ehret-Sabatier L , Bouet F , Goeldner M , Hirth C , Axelsen PH , Silman I , Sussman JL
Ref : Proceedings of the National Academy of Sciences of the United States of America , 90 :9031 , 1993
Abstract : Binding sites of Torpedo acetylcholinesterase (EC 3.1.1.7) for quaternary ligands were investigated by x-ray crystallography and photoaffinity labeling. Crystal structures of complexes with ligands were determined at 2.8-A resolution. In a complex with edrophonium, and quaternary nitrogen of the ligand interacts with the indole of Trp-84, and its m-hydroxyl displays bifurcated hydrogen bonding to two members of the catalytic triad, Ser-200 and His-440. In a complex with tacrine, the acridine is stacked against the indole of Trp-84. The bisquaternary ligand decamethonium is oriented along the narrow gorge leading to the active site; one quaternary group is apposed to the indole of Trp-84 and the other to that of Trp-279, near the top of the gorge. The only major conformational difference between the three complexes is in the orientation of the phenyl ring of Phe-330. In the decamethonium complex it lies parallel to the surface of the gorge; in the other two complexes it is positioned to make contact with the bound ligand. This close interaction was confirmed by photoaffinity labelling by the photosensitive probe 3H-labeled p-(N,N-dimethylamino)benzenediazonium fluoroborate, which labeled, predominantly, Phe-330 within the active site. Labeling of Trp-279 was also observed. One mole of label is incorporated per mole of AcChoEase inactivated, indicating that labeling of Trp-279 and that of Phe-330 are mutually exclusive. The structural and chemical data, together, show the important role of aromatic groups as binding sites for quaternary ligands, and they provide complementary evidence assigning Trp-84 and Phe-330 to the "anionic" subsite of the active site and Trp-279 to the "peripheral" anionic site.
ESTHER : Harel_1993_Proc.Natl.Acad.Sci.U.S.A_90_9031
PubMedSearch : Harel_1993_Proc.Natl.Acad.Sci.U.S.A_90_9031
PubMedID: 8415649
Gene_locus related to this paper: torca-ACHE

Title : Structure and functions of acetylcholinesterase and butyrylcholinesterase -
Author(s) : Massoulie J , Sussman JL , Bon S , Silman I
Ref : Prog Brain Res , 98 :139 , 1993
PubMedID: 8248501

Title : Three-dimensional structure of acetylcholinesterase and of its complexes with anticholinesterase drugs - Sussman_1993_Chem.Biol.Interact_87_187
Author(s) : Sussman JL , Harel M , Silman I
Ref : Chemico-Biological Interactions , 87 :187 , 1993
Abstract : Based on our recent X-ray crystallographic determination of the structure of acetylcholinesterase (AChE) from Torpedo californica, we can see for the first time, at atomic resolution, a protein binding pocket for the neurotransmitter, acetylcholine. It was found that the active site consists of a catalytic triad (S200-H440-E327) which lies close to the bottom of a deep and narrow gorge, which is lined with the rings of 14 aromatic amino acid residues. Despite the complexity of this array of aromatic rings, we suggested, on the basis of modelling which involved docking of the acetylcholine (ACh) molecule in an all-trans configuration, that the quaternary group of the choline moiety makes close contact with the indole ring of W84. In order to study the interaction of AChE with anticholinesterase drugs at the structural level, we have incorporated into the acetylcholinesterase crystals several different inhibitors, and have recently determined the 3-D structure of AChE:edrophonium and AChE:tacrine complexes. The crystal structures of both of these complexes are in good agreement with our model building of the ACh bound in the active site of AChE and indicate the interactions of these two drugs with the enzyme.
ESTHER : Sussman_1993_Chem.Biol.Interact_87_187
PubMedSearch : Sussman_1993_Chem.Biol.Interact_87_187
PubMedID: 8343975

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

Title : The alpha\/beta hydrolase fold - Ollis_1992_Prot.Engin_5_197
Author(s) : Ollis DL , Cheah E , Cygler M , Dijkstra B , Frolow F , Franken SM , Harel M , Remington SJ , Silman I , Schrag JD , Sussman JL , Verschueren KHG , Goldman A
Ref : Protein Engineering , 5 :197 , 1992
Abstract : We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.
ESTHER : Ollis_1992_Prot.Engin_5_197
PubMedSearch : Ollis_1992_Prot.Engin_5_197
PubMedID: 1409539

Title : G2-acetylcholinesterase is presynaptically localized in Torpedo electric organ - Eichler_1992_J.Neurocytol_21_707
Author(s) : Eichler J , Silman I , Anglister L
Ref : Journal of Neurocytology , 21 :707 , 1992
Abstract : In Torpedo electric organ, much of the acetylcholinesterase (AChE) is a globular dimer (G2), anchored to the plasma membrane via covalently attached phosphatidylinositol and selectively solubilized by a bacterial phosphatidylinositol-specific phospholipase C. While the structure of this form of the enzyme is well-established, the ultrastructural localization of G2-AChE is still unclear. Selective solubilization with phosphatidylinositol-specific phospholipase C was, therefore, combined with immunocytochemistry at the electron microscope level, in order to localize G2-AChE in electric organ of Torpedo ocellata. Thin sections of electric organ were labelled with antibodies raised against Torpedo AChE, followed by gold-conjugated second antibodies, before or after exposure to the phospholipase. For comparison, the location of AChE was examined using histochemical methods. We show that (1) immunolabelling is concentrated in the synaptic clefts between nerve terminals and the innervated face of the electrocyte; (2) this labelling co-localizes with AChE histochemical reaction products; and (3) prior exposure to the phospholipase causes a decrease in AChE-associated labelling. Quantitative analysis of immunolabelling in the synaptic clefts shows that the phospholipase treatment had reduced primary labelling at or adjacent to the presynaptic membrane. Together with our earlier biochemical and immunofluorescent evidence, these results support our previous assignment of a neuronal and synaptic localization for G2-AChE in Torpedo electric organ.
ESTHER : Eichler_1992_J.Neurocytol_21_707
PubMedSearch : Eichler_1992_J.Neurocytol_21_707
PubMedID: 1331340

Title : Chemical modification of Torpedo acetylcholinesterase by disulfides: appearance of a molten globule state - Dolginova_1992_Biochemistry_31_12248
Author(s) : Dolginova EA , Roth E , Silman I , Weiner L
Ref : Biochemistry , 31 :12248 , 1992
Abstract : Modification of Torpedo californica acetylcholinesterase (AChE) both by bis(1-oxy-2,2,5,5-tetramethyl-3-imidazolin-4-yl)disulfide (biradical) and by 4,4'-dithiopyridine, via a thiol-disulfide exchange reaction, was monitored by EPR and optical spectroscopy, respectively. Incubation with these reagents caused complete loss of enzymic activity. Treatment with glutathione of AChE modified by either of the two disulfides led to rapid release of the bound reagent with simultaneous regeneration of the single free thiol group of the enzyme. However, no concomitant recovery of catalytic activity was observed. SDS-PAGE showed that both the modified and demodified enzymes retained their structure as a disulfide-linked dimer. Circular dichroism revealed that modification of AChE by the disulfide agents with or without demodification by glutathione led to a complete disappearance of the ellipticity in the near-UV and to a much smaller decrease in ellipticity in the far-UV. The CD spectra observed are typical of the "molten globule" state of proteins. 1-Anilino-8-naphthalenesulfonate binding measurements and an enhanced susceptibility to trypsinolysis supported the supposition that chemical modification had transformed native AChE to a "molten globule".
ESTHER : Dolginova_1992_Biochemistry_31_12248
PubMedSearch : Dolginova_1992_Biochemistry_31_12248
PubMedID: 1333796

Title : Conversion of acetylcholinesterase to butyrylcholinesterase: modeling and mutagenesis - Harel_1992_Proc.Natl.Acad.Sci.U.S.A_89_10827
Author(s) : Harel M , Sussman JL , Krejci E , Bon S , Chanal P , Massoulie J , Silman I
Ref : Proceedings of the National Academy of Sciences of the United States of America , 89 :10827 , 1992
Abstract : Torpedo acetylcholinesterase (AcChoEase, EC 3.1.1.7) and human butyrylcholinesterase (BtChoEase, EC 3.1.1.8), while clearly differing in substrate specificity and sensitivity to inhibitors, possess 53% sequence homology; this permitted modeling human BtChoEase on the basis of the three-dimensional structure of Torpedo AcChoEase. The modeled BtChoEase structure closely resembled that of AcChoEase in overall features. However, six conserved aromatic residues that line the active-site gorge, which is a prominent feature of the AcChoEase structure, are absent in BtChoEase. Modeling showed that two such residues, Phe-288 and Phe-290, replaced by leucine and valine, respectively, in BtChoEase, may prevent entrance of butyrylcholine into the acyl-binding pocket. Their mutation to leucine and valine in AcChoEase, by site-directed mutagenesis, produced a double mutant that hydrolyzed butyrylthiocholine almost as well as acetylthiocholine. The mutated enzyme was also inhibited well by the bulky, BtChoEase-selective organophosphate inhibitor (tetraisopropylpyrophosphoramide, iso-OMPA). Trp-279, at the entrance of the active-site gorge in AcChoEase, is absent in BtChoEase. Modeling designated it as part of the "peripheral" anionic site, which is lacking in BtChoEase. The mutant W279A displayed strongly reduced inhibition by the peripheral site-specific ligand propidium relative to wild-type Torpedo AcChoEase, whereas inhibition by the catalytic-site inhibitor edrophonium was unaffected.
ESTHER : Harel_1992_Proc.Natl.Acad.Sci.U.S.A_89_10827
PubMedSearch : Harel_1992_Proc.Natl.Acad.Sci.U.S.A_89_10827
PubMedID: 1438284

Title : Site-directed mutagenesis of active-site-related residues in Torpedo acetylcholinesterase. Presence of a glutamic acid in the catalytic triad - Duval_1992_FEBS.Lett_309_421
Author(s) : Duval N , Bon S , Silman I , Sussman JL , Massoulie J
Ref : FEBS Letters , 309 :421 , 1992
Abstract : Site-directed mutagenesis was used to investigate the role of acidic amino acid residues close to the active site of Torpedo acetylcholinesterase. The recently determined atomic structure of this enzyme shows the conserved Glu-327, together with His-440 and Ser-200 as forming a catalytic triad, while the adjacent conserved Asp-326 points away from the active site. Transfection of appropriately mutated DNA into COS cells showed that the mutation of Asp-326----Asn had little effect on catalytic activity or the molecular forms expressed, suggesting no crucial structural or functional role for this residue. Mutation of Glu-327 to Gln or to Asp led to an inactive product. These results support the conclusions of the structural analysis for the two acidic residues.
ESTHER : Duval_1992_FEBS.Lett_309_421
PubMedSearch : Duval_1992_FEBS.Lett_309_421
PubMedID: 1355448

Title : Three Dimensional Structure of Acetylcholinesterase -
Author(s) : Sussman JL , Harel M , Silman I
Ref : In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases , (Shafferman, A. and Velan, B., Eds) Plenum Press, New York :95 , 1992
PubMedID:

Title : A Model of Butyrylcholinesterase Based on the X-Ray Structure of Acetylcholinesterase Indicates Differences in Specificity -
Author(s) : Harel M , Silman I , Sussman JL
Ref : In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases , (Shafferman, A. and Velan, B., Eds) Plenum Press, New York :189 , 1992
PubMedID:

Title : Site-Directed Mutagenesis of Functional Residues in Torpedo Acetylcholinesterase -
Author(s) : Silman I , Krejci E , Duval N
Ref : In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases , (Shafferman, A. and Velan, B., Eds) Plenum Press, New York :177 , 1992
PubMedID:

Title : Refined crystal structures of aged and non-aged organophosphoryl conjugates of gamma-chymotrypsin - Harel_1991_J.Mol.Biol_221_909
Author(s) : Harel M , Su CT , Frolow F , Ashani Y , Silman I , Sussman JL
Ref : Journal of Molecular Biology , 221 :909 , 1991
Abstract : "Aged" organophosphoryl conjugates of serine hydrolases differ from the corresponding "non-aged" conjugates in their striking resistance to nucleophilic reactivation. The refined X-ray structures of "aged" and "non-aged" organophosphoryl conjugates of gamma-chymotrypsin were compared in order to understand the molecular basis for this resistance of "aged" conjugates. "Aged" and "non-aged" crystalline organophosphoryl-gamma-chymotrypsin conjugates were obtained by prolonged soaking of native gamma-chymotrypsin crystals with appropriate organophosphates. Thus, a representative "non-aged" conjugate, diethylphosphoryl-gamma-chymotrypsin, was obtained by soaking native crystals with paraoxon (diethyl-p-nitrophenyl phosphate), and a closely related "aged" conjugate, monoisopropyl-gamma-chymotrypsin, was obtained by soaking with diisopropylphosphorofluoridate. In both crystalline conjugates, the refined structures clearly reveal a high occupancy of the active site by the appropriate organophosphoryl moiety within covalent bonding distance of Ser195 O gamma. Whereas in the "non-aged" conjugate both ethyl groups can be visualized clearly, in the putative "aged" conjugate, as expected, only one isopropyl group is present. There is virtually no difference between the "aged" and "non-aged" conjugates either with respect to the conformation of the polypeptide backbone as a whole or with respect to the positioning of the side-chains within the active site. In the "aged" conjugate, however, close proximity (2.6 A) of the negatively charged phosphate oxygen atom of the dealkylated organophosphoryl group to His57 N epsilon 2 indicates the presence of a salt bridge between these two moieties. In contrast, in the "non-aged" conjugate the DEP moiety retains its two alkyl groups; thus, lacking a negative oxygen atom, it does not enter into such a charge-charge interaction and its nearest oxygen atom is 3.6 A away from His57 N epsilon 2. It is suggested that steric constraints imposed by the salt bridge in the "aged" conjugate lie at the basis of its resistance to reactivation.
ESTHER : Harel_1991_J.Mol.Biol_221_909
PubMedSearch : Harel_1991_J.Mol.Biol_221_909
PubMedID: 1942036

Title : Effect of heat shock on acetylcholinesterase activity in chick muscle cultures - Eichler_1991_FEBS.Lett_293_16
Author(s) : Eichler J , Toker L , Silman I
Ref : FEBS Letters , 293 :16 , 1991
Abstract : The effect of heat shock was studied on the acetylcholinesterase activity of chick muscle primary cultures. In cultures transferred from 37 degrees C to 45 degrees C, a sharp drop in activity was followed by rapid spontaneous recovery. The time of onset of recovery resembled the time needed for expression of heat shock proteins. In cultures exposed to heat shock at 45 degrees C and allowed to recover at 37 degrees C, reappearance of acetylcholinesterase activity did not involve de novo protein synthesis since it was not prevented by cycloheximide. Our data raise the possibility of a role for heat shock proteins as molecular chaperones in rescuing heat-denaturing acetylcholinesterase.
ESTHER : Eichler_1991_FEBS.Lett_293_16
PubMedSearch : Eichler_1991_FEBS.Lett_293_16
PubMedID: 1959652

Title : Poster: Physicochemical and crystallographic studies on the stability and structure of aged and nonaged organophosphoryl conjugates of chymotrypsin -
Author(s) : Su CT , Steinberg N , Silman I , Harel M , Sussman JL , Grunwald J , Ashani Y
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 :274 , 1991
PubMedID:

Title : Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein - Sussman_1991_Science_253_872
Author(s) : Sussman JL , Harel M , Frolow F , Oefner C , Goldman A , Toker L , Silman I
Ref : Science , 253 :872 , 1991
Abstract : The three-dimensional structure of acetylcholinesterase from Torpedo californica electric organ has been determined by x-ray analysis to 2.8 angstrom resolution. The form crystallized is the glycolipid-anchored homodimer that was purified subsequent to solubilization with a bacterial phosphatidylinositol-specific phospholipase C. The enzyme monomer is an alpha/beta protein that contains 537 amino acids. It consists of a 12-stranded mixed beta sheet surrounded by 14 alpha helices and bears a striking resemblance to several hydrolase structures including dienelactone hydrolase, serine carboxypeptidase-II, three neutral lipases, and haloalkane dehalogenase. The active site is unusual because it contains Glu, not Asp, in the Ser-His-acid catalytic triad and because the relation of the triad to the rest of the protein approximates a mirror image of that seen in the serine proteases. Furthermore, the active site lies near the bottom of a deep and narrow gorge that reaches halfway into the protein. Modeling of acetylcholine binding to the enzyme suggests that the quaternary ammonium ion is bound not to a negatively charged "anionic" site, but rather to some of the 14 aromatic residues that line the gorge.
ESTHER : Sussman_1991_Science_253_872
PubMedSearch : Sussman_1991_Science_253_872
PubMedID: 1678899
Gene_locus related to this paper: torca-ACHE

Title : Phosphatidylinositol-specific phospholipase C induces biosynthesis of acetylcholinesterase via diacylglycerol in Schistosoma mansoni - Espinoza_1991_Eur.J.Biochem_195_863
Author(s) : Espinoza B , Silman I , Arnon R , Tarrab-Hazdai R
Ref : European Journal of Biochemistry , 195 :863 , 1991
Abstract : WWe have previously shown that two ectoenzymes, acetylcholinesterase (AChE) and alkaline phosphatase, are released from the surface and from particulate fractions of the parasite Schistosoma mansoni, by a phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of bacterial origin. Exposure to PtdIns-PLC not only removes large amounts of AChE from the surface of intact, viable Schistosoma in culture, but is accompanied by a concomitant increase in overall levels of AChE in the parasite. The same phenomenon is observed with PtdIns-PLC from two different bacterial sources; Staphylococcus aureus and Bacillus thuringiensis. The increase in AChE levels may be ascribed to de novo synthesis since exposure to PtdIns-PLC, in the presence of the protein-synthesis inhibitor cycloheximide, totally blocked the increase in AChE activity. Furthermore, PtdIns-PLC induced an increased incorporation of [35S]methionine into the AChE immunoprecipitated by a specific anti-AChE serum. This increase is selective for AChE, since total protein synthesis remained almost unchanged after PtdIns-PLC addition, and little or no effect was observed on the enzymatic activity of alkaline phosphatase, which is also glycophosphatidylinositol anchored. Since cleavage of the phosphatidylinositol anchor by PtdIns-PLC should liberate diacylglycerol, which may act as second messenger, we investigated the effect of exogenous diacylglycerols on the synthesis of AChE in S. mansoni. Three different diacylglycerols were tested as possible inducers of AChE activity in the parasite. Both 1-oleoyl-2-acetyl-sn-glycerol and 1,2-dimyristoyl-sn-glycerol were able to increase AChE activity by 35-40% at concentrations of 25 micrograms/ml. A higher concentration of 1,2-dioctanoyl-sn-glycerol (70 micrograms/ml) was needed to produce an equivalent effect. Moreover, addition of phorbol-12-myristate-13-acetate, together with the calcium ionophore A23187, produced a similar increase in AChE activity. Finally, polymixin B, a specific inhibitor of protein kinase C, partially blocked the increase in AChE activity induced by PtdIns-PLC. Our results suggest the involvement of glycophosphatidyl membrane-anchor breakdown products as putative second messengers in the parasite S. mansoni.
ESTHER : Espinoza_1991_Eur.J.Biochem_195_863
PubMedSearch : Espinoza_1991_Eur.J.Biochem_195_863
PubMedID: 1847873

Title : Poster: Effects of exposure to PIPL-C on AChE activity of Schistosoma mansoni -
Author(s) : Espinoza B , Silman I , Arnon R , Tarrab-Hazdai R
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 :50 , 1991
PubMedID:

Title : Poster: Immunocytochemical localization of phosphatidylinositol-anchored acetylcholinesterase in excitable membranes of Torpedo ocellata -
Author(s) : Eichler J , Silman I , Gentry MK , Anglister L
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 :49 , 1991
PubMedID:

Title : Structural Studies on Acetylcholinesterase from Torpedo californica -
Author(s) : Sussman JL , Harel M , Frolow F , Oefner C , Toker L , Silman I
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 :7 , 1991
PubMedID:

Title : Torpedo acetylcholinesterase is inactivated by thiol reagents - Steinberg_1990_Biochem.Int_21_1043
Author(s) : Steinberg N , Roth E , Silman I
Ref : Biochemistry International , 21 :1043 , 1990
Abstract : A number of sulphydryl reagents inhibit AChE of Torpedo california with pseudo-first-order kinetics, and inhibition can be retarded by quaternary ligands which bind at either the catalytic or peripheral anionic binding sites. Colorimetric determination with one of the inhibitory sulphydryl agents, 5,5'-dithiobis (2-nitrobenzoic acid), reveals the presence of a single thiol group per catalytic subunit; our data thus suggest that inhibition is achieved by reaction with the single free sulphydryl group of Cys231.
ESTHER : Steinberg_1990_Biochem.Int_21_1043
PubMedSearch : Steinberg_1990_Biochem.Int_21_1043
PubMedID: 1964378

Title : Immunocytochemical localization of phosphatidylinositol-anchored acetylcholinesterase in excitable membranes of Torpedo ocellata - Eichler_1990_Brain.Res.Molecular.Brain.Research_8_213
Author(s) : Eichler J , Silman I , Gentry MK , Anglister L
Ref : Brain Research Mol Brain Res , 8 :213 , 1990
Abstract : In Torpedo electric organ much of the acetylcholinesterase is a 'globular' dimer (G2), anchored to the plasma membrane via covalently attached phosphatidylinositol and solubilized by a bacterial phosphatidylinositol-specific phospholipase C. This suggested that selective solubilization with phosphatidylinositol-specific phospholipase C, coupled with immunocytochemistry, might be used to localize G2 acetylcholinesterase in excitable tissues of Torpedo. Cryostat sections of electric organ, electromotor nerve, electric lobe and back muscle from Torpedo ocellata were labelled, using three different antibody preparations to Torpedo acetylcholinesterase, followed by a fluorescent second antibody, before and after exposure to the phospholipase. Sites of innervation on electrocytes and myofibers were labelled selectively, as were motor and electromotor nerves. In all these cases labelling was substantially diminished by prior exposure to the phospholipase. The results support our previous assignment, based on biochemical evidence, for a neuronal and synaptic localization of the G2 acetylcholinesterase in Torpedo. Electric lobe acetylcholinesterase appears insensitive to the phospholipase treatment and lacks certain epitopes present in both electric organ and electromotor nerve enzyme. This suggests that substantial processing of the G2 form occurs concomitantly with its movement from the electric lobe into the electromotor nerve.
ESTHER : Eichler_1990_Brain.Res.Molecular.Brain.Research_8_213
PubMedSearch : Eichler_1990_Brain.Res.Molecular.Brain.Research_8_213
PubMedID: 2170799

Title : Conformational differences between aged and non-aged organophosphoryl conjugates of chymotrypsin -
Author(s) : Steinberg N , Grunwald J , Roth E , August R , Haas E , Ashani Y , Silman I
Ref : Prog Clin Biol Res , 289 :293 , 1989
PubMedID: 2726802

Title : Conformational differences between aged and nonaged pyrenebutyl-containing organophosphoryl conjugates of chymotrypsin as detected by optical spectroscopy - Steinberg_1989_Biochemistry_28_1248
Author(s) : Steinberg N , van der Drift AC , Grunwald J , Segall Y , Shirin E , Haas E , Ashani Y , Silman I
Ref : Biochemistry , 28 :1248 , 1989
Abstract : Homologous aged and nonaged fluorescent organophosphorus conjugates of alpha-chymotrypsin (Cht) were used in a comparative spectroscopic study of the conformation of their active sites, employing the pyrene group as the fluorescent probe. Steady-state fluorescence measurements showed that the quantum yield of the pyrene probe which is stoichiometrically attached to the active site is ca. 20% lower in the aged conjugate, pyrenebutyl-O-P(O)(O-)-Cht (PBP-Cht), than in the nonaged conjugate, pyrenebutyl-O-P-(O)(OC2H5)-Cht (PBEP-Cht). Furthermore, fluorescence decay data indicate that quenching is dynamic and is not caused by oxygen. These data, together with collisional quenching data, imply that quenching originates in an internal interaction of the fluorophore with a group within the protein. Thus, interaction of the pyrene moiety with the polypeptide chain is significantly stronger in the aged than in the nonaged conjugate, implying a different orientation of the fluorophore with respect to the protein. Circular dichroism measurements, which reflect the asymmetry of the bound pyrene in the ground state, as well as circularly polarized luminescence studies, which reflect its asymmetry in the excited state, also show that the relative configuration of the pyrene moiety and the polypeptide chain is significantly altered upon aging. Aged conjugates obtained by use of various fluorescenct organophosphates [pyrenebutyl-O-P(O)Cl2, pyrenebutyl-O-P(O)(p-nitrophenoxy)Cl, pyrenebutyl-O-P(O)(p-nitrophenoxy)2] exhibit similar spectroscopic features, thus substantiating the hypothesis that instantaneous aging, by use of pyrenebutyl-O-P(O)Cl2, and dynamic aging, by gradual removal of an aryloxy group, yield a similar product. This finding provides strong support for the formation of a P-O- moiety in the aged conjugates, since the only expected common product of the two processes is PB-O-P(O)(O-)-Cht. Formation of excimers of the pyrene-containing organophosphorylchymotrypsin conjugates at concentrations above 3 X 10(-6) M is also reported.
ESTHER : Steinberg_1989_Biochemistry_28_1248
PubMedSearch : Steinberg_1989_Biochemistry_28_1248
PubMedID: 2713362

Title : Purification and crystallization of a dimeric form of acetylcholinesterase from Torpedo californica subsequent to solubilization with phosphatidylinositol-specific phospholipase C - Sussman_1988_J.Mol.Biol_203_821
Author(s) : Sussman JL , Harel M , Frolow F , Varon L , Toker L , Futerman AH , Silman I
Ref : Journal of Molecular Biology , 203 :821 , 1988
Abstract : A dimeric form of acetylcholinesterase from Torpedo californica was purified to homogeneity by affinity chromatography subsequent to solubilization with a phosphatidylinositol-specific phospholipase C of bacterial origin. Bipyramidal crystals of the enzyme were obtained from solutions in polyethylene glycol 200. The crystals diffract to 2.0 A (1 A = 0.1 nm) resolution. They were found to be orthorhombic, space group P2221, with a = 163.4(+/- 0.2) A, b = 112.1(+/- 0.2) A, c = 81.3(+/- 0.1) A.
ESTHER : Sussman_1988_J.Mol.Biol_203_821
PubMedSearch : Sussman_1988_J.Mol.Biol_203_821
PubMedID: 2850366

Title : Visualization of collagenase-sensitive acetylcholinesterase in isolated cardiomyocytes and in heart tissue - Eghbali_1988_Cell.Tiss.Res_253_281
Author(s) : Eghbali M , Silman I , Robinson TF , Seifter S
Ref : Cell & Tissue Research , 253 :281 , 1988
Abstract : Previous studies have indicated that the asymmetric form of acetylcholinesterase (collagen-tailed) is localized in the basal lamina of the neuromuscular junction of skeletal muscle. The present study shows localization of the asymmetric acetylcholinesterase in the heart of the rat. Antiserum to 14 + 18 S acetylcholinesterase of the electric eel was raised in rabbits. The purified antibody did not react with collagen type I or laminin. Collagenase reduced the immunoreactivity of the enzyme with the purified antibody. Isolated cardiomyocytes and frozen sections of the heart were stained for acetylcholinesterase with the antibody. Diffuse immunofluorescence appeared over the surface of the cardiomyocytes. In the frozen sections, the immunofluorescence was most intense at the cell boundaries. These data suggest that collagenase-sensitive acetylcholinesterase in the heart is present in the myocytes and occurs in the vicinity of the basal lamina.
ESTHER : Eghbali_1988_Cell.Tiss.Res_253_281
PubMedSearch : Eghbali_1988_Cell.Tiss.Res_253_281
PubMedID: 2842053

Title : Acetylcholinesterase in Schistosoma mansoni is anchored to the membrane via covalently attached phosphatidylinositol - Espinoza_1988_Mol.Biochem.Parasitol_29_171
Author(s) : Espinoza B , Tarrab-Hazdai R , Silman I , Arnon R
Ref : Molecular & Biochemical Parasitology , 29 :171 , 1988
Abstract : Two enzymes, alkaline phosphatase and acetylcholinesterase (AChE), have been shown previously to be components of the surface of the trematode parasite Schistosoma mansoni. In this study we report that both these enzymes and other serine hydrolases are susceptible to release from the S. mansoni tegumental membrane by a phosphatidylinositol-specific phospholipase C (PIPLC) of bacterial origin. These data suggest that AChE and alkaline phosphatase of S. mansoni, as in higher organisms, are anchored to the membrane via covalently attached phosphatidylinositol. The release of AChE from the vesicular fraction of the parasite with PIPLC occurs in a concentration-dependent manner. Sucrose gradient centrifugation of the PIPLC-released AChE showed a single 8.3 S molecular form, similar to that observed for AChE solubilized by Triton X-100. PIPLC removed large amounts of AChE from the surface of intact schistosomula in culture, with no impairment of the viability of the parasite. In this case, an increase in the overall levels of AChE in the intact parasite was observed after addition of PIPLC.
ESTHER : Espinoza_1988_Mol.Biochem.Parasitol_29_171
PubMedSearch : Espinoza_1988_Mol.Biochem.Parasitol_29_171
PubMedID: 3137466

Title : A membrane-associated dimer of acetylcholinesterase from Xenopus skeletal muscle is solubilized by phosphatidylinositol-specific phospholipase C - Inestrosa_1988_Neurosci.Lett_90_186
Author(s) : Inestrosa NC , Fuentes ME , Anglister L , Futerman AH , Silman I
Ref : Neuroscience Letters , 90 :186 , 1988
Abstract : The susceptibility to phosphatidylinositol-specific phospholipase C of the membrane associated acetylcholinesterase (AChE) forms of Xenopus laevis skeletal muscle was examined. This treatment released almost all the detergent-soluble AChE species from muscle homogenates. Sucrose gradient analysis showed that the released acetylcholinesterase form corresponds to a hydrophilic G2 dimer, indicating that this dimer has a glycolipid anchoring domain which contains phosphatidylinositol.
ESTHER : Inestrosa_1988_Neurosci.Lett_90_186
PubMedSearch : Inestrosa_1988_Neurosci.Lett_90_186
PubMedID: 3412641

Title : Posttranslational modification as a means of anchoring acetylcholinesterase to the cell surface -
Author(s) : Silman I , Futerman AH
Ref : Biopolymers , 26 :S241 , 1987
PubMedID: 3555636

Title : Removal of covalently bound inositol from Torpedo acetylcholinesterase and mammalian alkaline phosphatases by deamination with nitrous acid. Evidence for a common membrane-anchoring structure - Low_1987_Biochem.J_241_615
Author(s) : Low MG , Futerman AH , Ackermann KE , Sherman WR , Silman I
Ref : Biochemical Journal , 241 :615 , 1987
Abstract : Our earlier evidence suggested that both acetylcholinesterase and alkaline phosphatase are anchored to the cell surface via covalently-attached phosphatidylinositol [Low, Futerman, Ferguson & Silman (1986) Trends Biochem. Sci. 11, 212-215]. We now present chemical data, based upon a nitrous acid deamination reaction, showing that in both proteins the phosphatidylinositol moiety is attached through a glycosidic linkage to a sugar residue bearing a free amino group.
ESTHER : Low_1987_Biochem.J_241_615
PubMedSearch : Low_1987_Biochem.J_241_615
PubMedID: 3593210

Title : Differential susceptibility to phosphatidylinositol-specific phospholipase C of acetylcholinesterase in excitable tissues of embryonic and adult Torpedo ocellata - Futerman_1987_Brain.Res_388_105
Author(s) : Futerman AH , Raviv D , Michaelson DM , Silman I
Ref : Brain Research , 388 :105 , 1987
Abstract : The ability of phosphatidylinositol-specific phospholipase C (PIPLC) to solubilize acetylcholinesterase (AChE) in the electromotor system of adult Torpedo ocellata and in the developing electric organ was examined. PIPLC solubilizes significant amounts of the membrane-bound G2 form of AChE throughout embryonic development of the electric organ, as it does in the adult electric organ, the AChE of which we have shown to contain covalently bound inositol in its membrane-anchoring domain. In the electromotor system of the mature fish, PIPLC solubilizes almost quantitatively the AChE dimer in the electromotor axon as in the electric organ itself, but the corresponding fraction in the electric lobe is almost totally resistant to the phospholipase. This finding implies that the covalently bound phosphatidylinositol is added concomitantly with axonal transport. A substantial part of the G2 form in back muscle is sensitive to PIPLC, whereas the G4 tetramer of Torpedo brain is completely resistant.
ESTHER : Futerman_1987_Brain.Res_388_105
PubMedSearch : Futerman_1987_Brain.Res_388_105
PubMedID: 3040164

Title : Modes of attachment of acetylcholinesterase to the surface membrane. - Silman_1987_Eur.J.Biochem_170_11
Author(s) : Silman I , Futerman AH
Ref : European Journal of Biochemistry , 170 :11 , 1987
Abstract : Acetylcholinesterase (AChE) occurs in multiple molecular forms differing in their quaternary structure and mode of anchoring to the surface membrane. Attachment is achieved by post-translational modification of the catalytic subunits. Two such mechanisms are described. One involves attachment to catalytic subunit tetramers, via disulfide bridges, of a collagen-like fibrous tail. This, in turn, interacts, primarily via ionic forces, with a heparin-like proteoglycan in the extracellular matrix. A second such modification involve the covalent attachment of a single phosphatidylinositol molecule at the carboxyl-terminus of each catalytic subunit polypeptide; the diacylglycerol moiety of the phospholipid serves to anchor the modified enzyme hydrophobically to the lipid bilayer of the plasma membrane. The detailed molecular structure of these two classes of acetylcholinesterase are discussed, as well as their biosynthesis and mode of anchoring.
ESTHER : Silman_1987_Eur.J.Biochem_170_11
PubMedSearch : Silman_1987_Eur.J.Biochem_170_11
PubMedID: 3319614

Title : Microtiter assay for acetylcholinesterase - Doctor_1987_Anal.Biochem_166_399
Author(s) : Doctor BP , Toker L , Roth E , Silman I
Ref : Analytical Biochemistry , 166 :399 , 1987
Abstract : A microtiter plate adaptation of the classical Ellman colorimetric procedure for measurement of acetylcholinesterase activity is described. This method permits use of an enzyme-linked immunosorbent assay plate reader for rapid analysis of multiple samples and is particularly suitable for analysis of acetylcholinesterase activity on sucrose gradients. The novel procedure is rapid and sensitive and does not require use of radioactive material.
ESTHER : Doctor_1987_Anal.Biochem_166_399
PubMedSearch : Doctor_1987_Anal.Biochem_166_399
PubMedID: 3434781

Title : Acetylcholinesterase of Schistosoma mansoni: purification and characterization - Goldlust_1986_J.Neurosci.Res_15_569
Author(s) : Goldlust A , Arnon R , Silman I , Tarrab-Hazdai R
Ref : Journal of Neuroscience Research , 15 :569 , 1986
Abstract : Larval acetylcholinesterase (acetylcholine acetylhydrolase) EC 3.1.3.7 of the trematode Schistosoma mansoni was characterized and purified by affinity chromatography. The enzyme was solubilized from sonicated cercarial tissue and showed a Km value of 1.83 mM and a Vmax value of 102 U/mg protein. It was characterized as a true AChE since it hydrolyses acetylthiocholine more than seven times faster than butyrylthiocholine, and since it was inhibited by high concentrations of substrate. The enzyme was purified by affinity chromatography on a Sepharose column of the inhibitor [N-(6-aminocaproyl-6-aminocaproyl)-m-aminophenyl] trimethyl ammonium. The purified enzyme eluted from the column by decamethonium bromide migrated as a single band of 500 kD on nondenaturing polyacrylamide gel electrophoresis (PAGE), whether stained for proteins or for enzymatic activity. Analysis by SDS-PAGE revealed two major polypeptide bands of 76 kD and 30 kD. By labeling the enzyme with 3H-DFP (di-isopropyl-fluorophosphate), the 30-kD polypeptide was shown to contain the active site of the enzyme, with an additional labeled band of 110 kD also being detected. On the basis of our data we suggest that the principal species of S. mansoni AChE is a tetramer of four subunit polypeptides each of MW ca. 110 kD which are not linked by disulfide bonds, and which are further cleaved into two fragments, one of MW 76,000 and one of MW 30,000, the latter bears the active site.
ESTHER : Goldlust_1986_J.Neurosci.Res_15_569
PubMedSearch : Goldlust_1986_J.Neurosci.Res_15_569
PubMedID: 3723610

Title : Butyrylcholinesterase: inhibition by arsenite, fluoride, and other ligands, cooperativity in binding - Page_1985_Mol.Pharmacol_27_437
Author(s) : Page JD , Wilson IB , Silman I
Ref : Molecular Pharmacology , 27 :437 , 1985
Abstract : Arsenite is a quasi-irreversible inhibitor of human serum butyrylcholinesterase with a dissociation constant of 0.129 mM at pH 7.4, 25 degrees, 0.067 M phosphate, mu = 0.17 M. The inhibition process is second order with a rate constant of 340 M-1 min-1. The first order rate of dissociation, 0.044 min-1, is unaffected by fluoride but is decreased by substrate. The binding of arsenite and fluoride, as determined by the effect of fluoride on the apparent arsenite-enzyme dissociation constant, is highly anticooperative and may be mutually exclusive. The fluoride-enzyme dissociation constant determined from these experiments is 0.90 mM. The binding of a number of other substances, such as dibucaine, is markedly anticooperative with arsenite binding. The binding of some of these substances is positively cooperative with fluoride binding. The effect can be large; procainamide binds 17 times more strongly in the presence of fluoride. Similarly, the mutual binding of benzoylcholine as substrate and fluoride is cooperative, 30-fold, butyrylthiocholine and fluoride, 21-fold, propionylthiocholine and fluoride, 8.3-fold, and acetylthiocholine and fluoride, only 1.8-fold.
ESTHER : Page_1985_Mol.Pharmacol_27_437
PubMedSearch : Page_1985_Mol.Pharmacol_27_437
PubMedID: 3982389

Title : Physicochemical behaviour and structural characteristics of membrane-bound acetylcholinesterase from Torpedo electric organ. Effect of phosphatidylinositol-specific phospholipase C - Futerman_1985_Biochem.J_226_369
Author(s) : Futerman AH , Fiorini RM , Roth E , Low MG , Silman I
Ref : Biochemical Journal , 226 :369 , 1985
Abstract : Quantitative solubilization of the phospholipid-associated form of acetylcholinesterase (AChE) from Torpedo electric organ can be achieved in the absence of detergent by treatment with phosphatidylinositol-specific phospholipase C (PIPLC) from Staphylococcus aureus [Futerman, Low & Silman (1983) Neurosci. Lett. 40, 85-89]. The sedimentation coefficient on sucrose gradients of AChE solubilized in detergents (DSAChE) varies with the detergent employed. However, the coefficient of AChE directly solubilized by PIPLC is not changed by detergents. Furthermore, PIPLC can abolish the detergent-sensitivity of the sedimentation coefficient of DSAChE purified by affinity chromatography, suggesting that one or more molecules of phosphatidylinositol (PI) are co-solubilized with DSAChE and remain attached throughout purification. DSAChE binds to phospholipid liposomes, whereas PIPLC-solubilized AChE and DSAChE treated with PIPLC do not bind even to liposomes containing PI. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows that PIPLC-solubilized AChE, like unmodified DSAChE, is a catalytic subunit dimer; electrophoresis in the presence of reducing agent reveals no detectable difference in the Mr of the catalytic subunit of unmodified DSAChE, of AChE solubilized by PIPLC and of AChE solubilized by Proteinase K. The results presented suggest that DSAChE is anchored to the plasma membrane by one or more PI molecules which are tightly attached to a short amino acid sequence at one end of the catalytic subunit polypeptide.
ESTHER : Futerman_1985_Biochem.J_226_369
PubMedSearch : Futerman_1985_Biochem.J_226_369
PubMedID: 2986594

Title : Identification of covalently bound inositol in the hydrophobic membrane-anchoring domain of Torpedo acetylcholinesterase - Futerman_1985_Biochem.Biophys.Res.Commun_129_312
Author(s) : Futerman AH , Low MG , Ackermann KE , Sherman WR , Silman I
Ref : Biochemical & Biophysical Research Communications , 129 :312 , 1985
Abstract : The hydrophobic, membrane-bound form of Torpedo acetylcholinesterase is specifically solubilized by a phosphatidylinositol-specific phospholipase C, suggesting that acetylcholinesterase is bound to the membrane via a direct and specific interaction with phosphatidylinositol (Futerman et al., Biochem. J. (1985) 226, 369-377). Here we demonstrate the presence of covalently bound inositol in the membrane-anchoring domain of purified Torpedo acetylcholinesterase. Upon removal of this domain, levels of inositol are reduced to only 15-20% of those found in the intact enzyme.
ESTHER : Futerman_1985_Biochem.Biophys.Res.Commun_129_312
PubMedSearch : Futerman_1985_Biochem.Biophys.Res.Commun_129_312
PubMedID: 4004881

Title : Solubilization of membrane-bound acetylcholinesterase by a phosphatidylinositol-specific phospholipase C - Futerman_1985_J.Neurochem_45_1487
Author(s) : Futerman AH , Low MG , Michaelson DM , Silman I
Ref : Journal of Neurochemistry , 45 :1487 , 1985
Abstract : Phosphatidylinositol-specific phospholipase C (PIPLC) quantitatively solubilizes acetylcholinesterase (AChE) from purified synaptic plasma membranes and intact synaptosomes of Torpedo ocellata electric organ. The solubilized AChE migrates as a single peak of sedimentation coefficient 7.0S upon sucrose gradient centrifugation, corresponding to a subunit dimer. The catalytic subunit polypeptide of AChE is the only polypeptide detectably solubilized by PIPLC. This selective removal of AChE does not affect the amount of acetylcholine released from intact synaptosomes upon K+ depolarization. PIPLC also quantitatively solubilizes AChE from the surface of intact bovine and rat erythrocytes, but only partially solubilizes AChE from human and mouse erythrocytes. The AChE released from rat and human erythrocytes by PIPLC migrates as a approximately 7S species on sucrose gradients, corresponding to a catalytic subunit dimer. PIPLC does not solubilize particulate AChE from any of the brain regions examined of four mammalian species. Several other phospholipases tested, including a nonspecific phospholipase C from Clostridium welchii, fail to solubilize AChE from Torpedo synaptic plasma membranes, rat erythrocytes, or rat striatum.
ESTHER : Futerman_1985_J.Neurochem_45_1487
PubMedSearch : Futerman_1985_J.Neurochem_45_1487
PubMedID: 4045459

Title : Arrhenius plots of acetylcholinesterase activity in mammalian erythrocytes and in Torpedo electric organ. Effect of solubilization by proteinases and by a phosphatidylinositol-specific phospholipase C - Barton_1985_Biochem.J_231_237
Author(s) : Barton PL , Futerman AH , Silman I
Ref : Biochemical Journal , 231 :237 , 1985
Abstract : The temperature-dependence of the catalytic activity of acetylcholinesterase (AChE) from rat erythrocyte-ghost membranes and from Torpedo electric-organ membranes was examined. In the case of rat erythrocyte AChE, a non-linear Arrhenius plot was observed both before and after solubilization by a phosphatidylinositol-specific phospholipase C or by proteinase treatment. Similarly, no significant differences were observed in Arrhenius plots of Torpedo electric-organ AChE before or after solubilization. These results support our suggestion that the catalytic subunit of AChE does not penetrate deeply into the lipid bilayer of the plasma membrane and also suggest that care must be taken in ascribing break points in Arrhenius plots of membrane-bound enzymes to changes in their lipid environment.
ESTHER : Barton_1985_Biochem.J_231_237
PubMedSearch : Barton_1985_Biochem.J_231_237
PubMedID: 3904734

Title : Solubilization of membrane-bound acetylcholinesterase by a phosphatidylinositol-specific phospholipase C: enzymatic and physicochemical studies -
Author(s) : Futerman AH , Fiorini RM , Roth E , Michaelson DM , Low MG , Silman I
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter :99 , 1984
PubMedID:

Title : Ontogenic and agranulation-induced alterations in cholinesterases and in cholinesterase mRNA in the rodent cerebellum -
Author(s) : Parvari R , Silman I , Soreq H
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter :219 , 1984
PubMedID:

Title : Poster 9. Solubilisation of acetylchoinesterase from Torpedo electric organ by a phosphatidylinositol-specific phospholipase C -
Author(s) : Futerman AH , Low MG , Silman I
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter , 1984
PubMedID:

Title : Poster 19. Molecular forms of the cholinesterases Inside and outside endplates in. slow-tonic chicken muscle fibers -
Author(s) : Jedrzejczyk J , Silman I , Barnard EA
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter , 1984
PubMedID:

Title : Characterization of activities and forms of cholinesterases in human primary brain tumors - Razon_1984_Exp.Neurol_84_681
Author(s) : Razon N , Soreq H , Roth E , Bartal A , Silman I
Ref : Experimental Neurology , 84 :681 , 1984
Abstract : The activities and molecular forms of cholinesterases were studied in a collection of primary brain tumors consisting of primarily gliomas and meningiomas, together with samples of forebrain taken postmortem from patients suffering from diseases unrelated to the nervous system. Both types of tumors, as well as normal forebrain, contained substantial amounts of cholinesterase activity and some gliomas contained exceptionally high levels. In both normal forebrain and meningiomas, acetylcholinesterase (acetylcholine hydrolase; EC 3.1.1.7) accounted for almost all the cholinesterase activity, but in almost all gliomas elevated pseudocholinesterase (acylcholine acylhydrolase; EC 3.1.1.8) could be detected. The cholinesterase activity of both normal forebrain and gliomas migrated on sucrose gradients as a major component of 10-11 S together with a minor component of 4-5 S. In meningiomas a light (4.5 S) form was the principal component.
ESTHER : Razon_1984_Exp.Neurol_84_681
PubMedSearch : Razon_1984_Exp.Neurol_84_681
PubMedID: 6723888

Title : Molecular forms of acetylcholinesterase in synaptic and extrasynaptic regions of avian tonic muscle - Jedrzejczyk_1984_Neurosci.Lett_46_283
Author(s) : Jedrzejczyk J , Silman I , Lai J , Barnard EA
Ref : Neuroscience Letters , 46 :283 , 1984
Abstract : Molecular forms of acetylcholinesterase and pseudocholinesterase were analyzed directly in the micro-dissected individual endplates of a slow-tonic chicken muscle. The major form in the endplate is the L2(6.5 S) form, while the collagen-tailed H2c (20 S) form, normally considered to be the synaptic form, is a very minor component, in contrast to its predominance at the chicken fast-twitch fibre endplate. The same is true for pseudocholinesterase at these endplates. Outside the tonic fibre endplates the same forms occur as at the endplates, but at a very much lower concentration. The enzyme at the tonic fibre endplate cannot be attached to the basal lamina by a collagen tail, but appears to have a hydrophobic attachment. Acetylcholinesterase is functional at tonic fibre endplates, but the absence of the collagen-tailed form may account for the lower efficiency of the enzymic removal of acetylcholine there.
ESTHER : Jedrzejczyk_1984_Neurosci.Lett_46_283
PubMedSearch : Jedrzejczyk_1984_Neurosci.Lett_46_283
PubMedID: 6738921

Title : A hydrophobic dimer of acetylcholinesterase from Torpedo californica electric organ is solubilized by phosphatidylinositol-specific phospholipase C - Futerman_1983_Neurosci.Lett_40_85
Author(s) : Futerman AH , Low MG , Silman I
Ref : Neuroscience Letters , 40 :85 , 1983
Abstract : A dimeric form of acetylcholinesterase from the electric organ of Torpedo californica was solubilized by phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. The solubilized enzyme had a sedimentation coefficient of 7.3S which was not modified by detergents. The high salt-soluble asymmetric forms of acetylcholinesterase were not solubilized by the phospholipase. Our data suggest that the hydrophobic dimer of acetylcholinesterase may be associated with the plasma membrane through a specific interaction involving phosphatidylinositol.
ESTHER : Futerman_1983_Neurosci.Lett_40_85
PubMedSearch : Futerman_1983_Neurosci.Lett_40_85
PubMedID: 6633970

Title : Biosynthesis of acetylcholinesterase in rat brain and Torpedo electric organ is directed by scarce mRNA species -
Author(s) : Soreq H , Parvari R , Silman I
Ref : Progress in Brain Research , 58 :107 , 1983
PubMedID: 6635185

Title : Novel pyrene-containing organophosphates as fluorescent probes for studying aging-induced conformational changes in organophosphate-inhibited acetylcholinesterase -
Author(s) : Amitai G , Ashani Y , Gafni A , Silman I
Ref : Biochemistry , 21 :2060 , 1982
PubMedID: 7093230

Title : Molecular forms of acetylcholinesterase and pseudocholinesterase in chicken skeletal muscles: their distribution and change with muscular dystrophy - Barnard_1982_Rep.Nutr.Dev_22_261
Author(s) : Barnard EA , Lyles JM , Silman I , Jedrzejczyk J , Barnard PJ
Ref : Reproduction, Nutrition, Developpement , 22 :261 , 1982
Abstract : Chicken muscles offer several significant advantages for the use of cholinesterase as a marker of nerve-muscle interactions. A series of molecular forms of chicken muscle acetylcholinesterase (AChE), and likewise of pseudocholinesterase (psi ChE), has been defined. The form of AChE inside the endplates of fast-twitch muscle is H2c (20 S), with a collagenous tail. The same is true for psi ChE. The changes in these forms in the muscle with embryonic development, with muscle fibre-type composition and under the influence of inherited muscular dystrophy, are described quantitatively.
ESTHER : Barnard_1982_Rep.Nutr.Dev_22_261
PubMedSearch : Barnard_1982_Rep.Nutr.Dev_22_261
PubMedID: 7156481

Title : Comparison of the molecular forms of the cholinesterases in tissues of normal and dystrophic chickens - Lyles_1982_J.Neurochem_38_1007
Author(s) : Lyles JM , Silman I , Di Giamberardino L , Couraud JY , Barnard EA
Ref : Journal of Neurochemistry , 38 :1007 , 1982
Abstract : The levels and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and pseudocholinesterase (psiChE, EC 3.1.1.8) were examined in various skeletal muscles, cardiac muscles, and neural tissues from normal and dystrophic chickens. The relative amount of the heavy (Hc) form of AChE in mixed-fibre-type twitch muscles varies in proportion to the percentage of glycolytic fast-twitch fibres. Conversely, muscles with higher levels of oxidative fibres (i.e., slow-tonic oxidative-glycolytic fast-twitch, or oxidative slow-twitch) have higher proportions of the light (L) form of AChE. The effects of dystrophy on AChE and psiChE are more severe in muscles richer in glycolytic fast-twitch fibres (e.g., pectoral or posterior latissimus dorsi, PLD); there is no alteration of AChE or psiChE in a slow-tonic muscle. In the pectoral of PLD muscles from older dystrophic chickens, however, the AChE forms revert to a normal distribution while the pesChE pattern remains abnormal. Muscle psiChE is sensitive to collagenase in a similar way as is AChE, thus apparently having a similar tailed structure. Unlike skeletal muscle, cardiac muscle has very high levels of psiChE, present mainly as the L form; AChE is present mainly as the medium (M) form, with smaller amounts of L and Hc. The latter pattern of AChE forms resembles that seen in several neural tissues examined. No alterations in AChE or psiChE were found in cardiac or neural tissues from dystrophic chickens.
ESTHER : Lyles_1982_J.Neurochem_38_1007
PubMedSearch : Lyles_1982_J.Neurochem_38_1007
PubMedID: 7062026

Title : Biosynthesis and secretion of catalytically active acetylcholinesterase in Xenopus oocytes microinjected with mRNA from rat brain and from Torpedo electric organ - Soreq_1982_Proc.Natl.Acad.Sci.U.S.A_79_830
Author(s) : Soreq H , Parvari R , Silman I
Ref : Proceedings of the National Academy of Sciences of the United States of America , 79 :830 , 1982
Abstract : A novel technique was developed for monitoring the level of the mRNA species that direct the synthesis of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7), using microinjected Xenopus oocytes as a translation system. When injected with poly(A)-containing RNA from whole rat brain or rat cerebellum and from electric organ of Torpedo ocellata, Xenopus oocytes synthesize and secrete catalytically active cholinesterase. The newly synthesized enzyme, which is mostly secreted into the oocytes incubation medium, appears to be primarily AcChoEase because it is inhibited by the specific inhibitor BW 284C51. The new enzymatic activity can be detected after injection of as little as 12.5 ng of poly(A)-containing RNA per oocyte, and there is a linear dependence of the oocytes' ability to form AcChoEase on the amount of injected RNA. The AcChoEase mRNA displays a tau 1/2 of about 10 +/- 3 hr in injected oocytes. The abundance of AcChoEase mRNA in the total nonfractionated mRNA injected was calculated to be ca. 1 x 10(-5), a value similar to the level of AcChoEase protein determined in rat brain. The combination of the high turnover number of AcChoEase, the efficiency of the oocyte system, and the sensitivity of the assay used thus permit the accurate monitoring of the scarce mRNA species that direct the synthesis of this enzyme.
ESTHER : Soreq_1982_Proc.Natl.Acad.Sci.U.S.A_79_830
PubMedSearch : Soreq_1982_Proc.Natl.Acad.Sci.U.S.A_79_830
PubMedID: 6950429

Title : In vivo modulation of the number of muscarinic receptors in rat brain by cholinergic ligands - Ben-Barak_1981_Eur.J.Pharmacol_74_73
Author(s) : Ben-Barak J , Gazit H , Silman I , Dudai Y
Ref : European Journal of Pharmacology , 74 :73 , 1981
Abstract : Administration of the muscarinic agonist oxotremorine led to a decrease in the number of muscarinic receptors, as determined by specific binding of [3H]quinuclidinyl benzilate ([3H]QNB), in several rat brain regions both during development and at maturity. In contrast, administration of the muscarinic antagonist scopolamine led to an increase in the number of [3H]QNB-binding sites in various brain regions. Scopolamine also prevented the decrease in the number of [3H]QNB-binding sites induced by administration of an organophosphorus drug. The results are compatible with the hypothesis that the number of brain muscarinic receptors, or at least of a sub-class of them, is regulated by their transmitter.
ESTHER : Ben-Barak_1981_Eur.J.Pharmacol_74_73
PubMedSearch : Ben-Barak_1981_Eur.J.Pharmacol_74_73
PubMedID: 7318895

Title : Molecular forms of the cholinesterases inside and outside muscle endplates - Jedrzejczyk_1981_Biosci.Rep_1_45
Author(s) : Jedrzejczyk J , Silman I , Lyles JM , Barnard EA
Ref : Bioscience Reports , 1 :45 , 1981
Abstract : Individual endplates were micro-dissected from chicken fast-twitch muscle, and the molecular forms of acetylcholinesterase and of pseudocholinesterase therein, identified by their sedimentation coefficients, were analysed directly. The forms actually present at the endplate, and those that are non-synaptic, were established. This analysis was also extended to muscle of the chicken with inherited muscular dystrophy, showing altered distributions of these forms.
ESTHER : Jedrzejczyk_1981_Biosci.Rep_1_45
PubMedSearch : Jedrzejczyk_1981_Biosci.Rep_1_45
PubMedID: 7284574

Title : New fluorescent organophosphates as probes for studying aging-induced conformational changes in inhibited acetylcholinesterase - Amitai_1980_Neurochem.Int_2C_199
Author(s) : Amitai G , Ashani Y , Gafni A , Silman I
Ref : Neurochem Int , 2C :199 , 1980
Abstract : Aging of organophosphoryl-acetylcholinesterase (AChE) conjugates, involving dealkylation of the bound organophosphoryl group, renders AChE resistant to reactivation by 2-pyridinealdoxime methiodide (2-PAM). The fluorescent organophosphates 1-pyrenebutyl ethylphosphorochloridate (PBEPC) and 1-pyrenebutylphosphorodichloridate (PBPDC) react stoichiometrically with purified electric eel AChE. PBEPC forms a non-aged AChE conjugate readily reactivated by 2-PAM; PBPDC forms an aged conjugate which cannot be reactivated. There is no difference in the wavelengths of excitation and emission maxima between the aged and non-aged AChE conjugates. However, the fluorescence quantum yield of pyrene in the non-aged conjugate is reduced by ca. 50% compared to the aged conjugate and from the shortening of the fluorescence decay time in the non-aged conjugate, it is concluded that the quenching is primarily dynamic. It is suggested that in the aged conjugate the organophosphoryl moiety is less accessible to the external medium than in the non-aged conjugate.
ESTHER : Amitai_1980_Neurochem.Int_2C_199
PubMedSearch : Amitai_1980_Neurochem.Int_2C_199
PubMedID: 20487784

Title : Changes in the levels and forms of cholinesterases in the blood plasma of normal and dystrophic chickens -
Author(s) : Lyles JM , Barnard EA , Silman I
Ref : Journal of Neurochemistry , 34 :978 , 1980
PubMedID: 7359144

Title : Electric eel acetylcholinesterase: a multisubunit enzyme containing a collagen tail - Silman_1980_Monogr.Neural.Sci_7_55
Author(s) : Silman I , Anglister L
Ref : Monographs in Neural Sciences , 7 :55 , 1980
Abstract : Molecular forms of acetylcholinesterase (AChE) in fresh electric organ tissue are elongated structures in which a multisubunit head containing the catalytic sites is attached to a fibrous tail. The principal form, 18S AChE, is of MW ca. 1,100,000 and aggregates reversibly at low ionic strength. Trypsin converts it to an 11S globular tetramer devoid of the tail and lacking the capacity to aggregate reversibly in low salt. Amino acid analysis, collagenase and pepsin digestion and immunological techniques were utilized to demonstrate that the fibrous tail of the elongated forms of AChE is a collagen triple helix. The distal portion of the tail contains a region responsible for the capacity for aggregation at low ionic strength. This latter property may be related to the postulated role of the tail in anchoring AChE to the fibrillar matrix of the basal lamina.
ESTHER : Silman_1980_Monogr.Neural.Sci_7_55
PubMedSearch : Silman_1980_Monogr.Neural.Sci_7_55
PubMedID: 6262636

Title : Fluorescent organophosphates: novel probes for studying aging-induced conformational changes in inhibited acetylcholinesterase and for localization of cholinesterase in nervous tissue - Amitai_1980_Monogr.Neural.Sci_7_70
Author(s) : Amitai G , Ashani Y , Shahar A , Gafni A , Silman I
Ref : Monographs in Neural Sciences , 7 :70 , 1980
Abstract : Aging of acetylcholinesterase (AChE) inhibited by certain organophosphates such as diisopropylfluorophosphate apparently involves dealkylation of the bound organophosphoryl moiety; this renders the inactive enzyme resistant to reactivation by quaternary oximes such as 2-pyridinealdoxime methiodide (2-PAM) which are used in therapy of organophosphate intoxication. The fluorescent pyrenyl organophosphates synthesized in this study were designed to detect putative conformational changes which might explain this resistance. The following inhibitors: 1-pyrenebutyl phosphorodichloride (PBPDC), 1-pyrenebutyl ethylphosphorochloridate (PBEPC), and 1-pyrenebutyl ethylphosphorofluoridate (PBEPF), react specifically with purified electric eel AChE (ki = 10(6)-10(7) M-1 min-1). AChE inhibited by PBEPC and PBEPF was readily reactivated by 2-PAM, while enzyme inhibited PBPDC could not be reactivated. Conjugates were prepared of both PBEPC and PBPDC with AChE, each containing one molecule of florophore per catalytic subunit. Thus two stoichiometric conjugates, PBEP-AChE (non-aged) and POBP-AChE (aged), were obtained. The two complexes exhibited identical absorption spectra, but differed in their steady-state fluorescence spectra. Although the wave-lenths of the excitation and emission spectra were similar, the pyrene fluorescence of the non-aged conjugate was ca. 50% quenched relative to the aged conjugate. Nanosecond fluorescence decay studies revealed two principal lifetime components of pyrene fluorescence. Both were longer for the aged (PBP-AChE) than for the non-aged (PBEP-AChE) conjugate and revealed a ca. 50% lower quantum yield for the non-aged as compared to the aged conjugate. A possible interpretation for these results is that in the aged conjugate the organophosphoryl moiety is less acessible to the external medium. Measurement of quenching of pyrene fluorescence in the aged and non-aged conjugates by the peripheral anionic site ligand propidium also indicated marked conformational differences between the two conjugates, and circular polarization of luminescence measurements revealed that propidium itself induced a substantial conformational change in both conjugates. Fluorescence lifetime measurements revealed that whereas propidium had little effect on the decay parameters for the non-aged conjugate it caused a decrease in lifetime and in relative quantum yield for the aged conjugate. PBEPF virtually eliminated cholinesterase activity in dissociated cord and brain cultures. Fluorescence microscopy reveals fine green fluorescent grains distinctly located throughout many neurons and glia. Labelling is much more pronounced in larger and older neurons. No specific fluorescence could be detected in cultures preincubated with nonfluorescent organophosphates.
ESTHER : Amitai_1980_Monogr.Neural.Sci_7_70
PubMedSearch : Amitai_1980_Monogr.Neural.Sci_7_70
PubMedID: 7015117

Title : The induction of ion channels through excitable membranes by acetylcholinesterase -
Author(s) : Kaufmann K , Silman I
Ref : Naturwissenschaften , 67 :608 , 1980
PubMedID: 6262662

Title : Immunological cross-reactivity between electric-eel acetylcholinesterase and rat-tail-tendon collagen - Anglister_1979_Eur.J.Biochem_94_25
Author(s) : Anglister L , Tarrab-Hazdai R , Fuchs S , Silman I
Ref : European Journal of Biochemistry , 94 :25 , 1979
Abstract : Immunological cross-reactivity between acetylcholinesterase from the electric organ of the electric eel and rat tail tendon collagen was examined both on the cellular and humoral levels. 1. Guinea pigs immunized with rat tail tendon collagen displayed a strong delayed-type skin reaction when tested with the elongated acetylcholinesterase preparation (i.e. 14-S + 18-S molecular forms). However, when the glubular 11-S enzyme was tested, almost no cross-reactivity was obtained. Similarly, guinea pigs immunized with 14-S + 18-S preparation exhibited skin sensitization to rat tail tendon collagen. 2. Using a radioimmunoassay, it was observed that 125I-labeled 14-S + 18-S acetylcholinesterase binds efficiently to rabbit antiserum elicited against rat tail tendon collagen, whereas 125I-labeled 11-S enzyme does not bind at all to this antiserum. Similar results were obtained by passive hemagglutination assay. The experiments suggest that 14-S + 18-S acetylcholinesterase, but not 11-S enzyme, which is devoid of the tail structure, has antigenic determinants in common with collagen from rat tail tendon.
ESTHER : Anglister_1979_Eur.J.Biochem_94_25
PubMedSearch : Anglister_1979_Eur.J.Biochem_94_25
PubMedID: 436842

Title : Developmental changes in levels and forms of cholinesterases in muscles of normal and dystrophic chickens -
Author(s) : Lyles JM , Silman I , Barnard EA
Ref : Journal of Neurochemistry , 33 :727 , 1979
PubMedID: 479887

Title : Administration of an organophosphate causes a decrease in muscarinic receptor levels in rat brain -
Author(s) : Gazit H , Silman I , Dudai Y
Ref : Brain Research , 174 :351 , 1979
PubMedID: 487134

Title : Parallel regulation of acetylcholinesterase and pseudocholinesterase in normal, denervated and dystrophic chicken skeletal muscle -
Author(s) : Silman I , Di Giamberardino L , Lyles L , Couraud JY , Barnard EA
Ref : Nature , 280 :160 , 1979
PubMedID: 552605

Title : Inactivation of electric eel acetylcholinesterase by acylation with N-hydroxysuccinimide esters of amino acid derivatives -
Author(s) : Blumberg S , Silman I
Ref : Biochemistry , 17 :1125 , 1978
PubMedID: 629951

Title : Intrinsic forms of acetylcholinesterase in skeletal muscle -
Author(s) : Silman I , Lyles JM , Barnard EA
Ref : FEBS Letters , 94 :166 , 1978
PubMedID: 700129

Title : Molecular structure of elongated forms of electric eel acetylcholinesterase -
Author(s) : Anglister L , Silman I
Ref : Journal of Molecular Biology , 125 :293 , 1978
PubMedID: 215774

Title : Effects of quaternary ligands on the inhibition of acetylcholinesterase by arsenite - Wilson_1977_Biochemistry_16_2701
Author(s) : Wilson IB , Silman I
Ref : Biochemistry , 16 :2701 , 1977
Abstract : Arsenite inhibits acetylcholinesterase in a second-order reaction. The rate and equilibrium constants depend upon pH and have values on the order of 10(2) M-1 min-1 and 10(5) M (dissociation), respectively. Some quaternary ammonium ligands completely block the arsenite inhibition of the enzyme, others decrease the rate of the reaction and some, notably pyridine-2 aldoxime methiodide, greatly accelerate the rate of the reaction, up to 220-fold. Accelerators may bind at a separate enzyme site distinct form the anionic site involved in substrate binding. Although the kinetic data are consistent with a covalent reaction between arsenite and acetylcholinesterase, chemical evidence excludes the involvement of sulfhydryl groups which are usually implicated in arsenite inhibition.
ESTHER : Wilson_1977_Biochemistry_16_2701
PubMedSearch : Wilson_1977_Biochemistry_16_2701
PubMedID: 19036

Title : Characterization of membranes obtained from electric organ of the electric eel by sucrose gradient fractionation and by microdissection -
Author(s) : Rosenberg P , Silman I , Ben-David E , De Vries A , Condrea E
Ref : Journal of Neurochemistry , 29 :561 , 1977
PubMedID: 142822

Title : Acetylcholinesterase: structure and activity of a membrane-bound enzyme -
Author(s) : Silman I , Dudai Y
Ref : Advances in Biological & Medical Physics , 16 :223 , 1977
PubMedID: 45171

Title : Appearance and disappearance of acetycholine receptor during differentiation of chick skeletal muscle in vitro - Prives_1976_Cell_7_543
Author(s) : Prives J , Silman I , Amsterdam A
Ref : Cell , 7 :543 , 1976
Abstract : During differentiation of embryonic chick skeletal muscle in culture, elaboration of acetylcholine receptor (AChR) and acetylcholinesterase occurs shortly after myoblast fusion. During further development, AChR was found to decrease markedly on the myotube surface, while acetylcholinesterase continued to increase. Surface distribution of AChR, as followed by autoradiography using 125I-alpha-bungarotoxin, was homogeneous in newly fused myotubes. With further differentiation, clusters of AChR appeared on the surface of the myotubes, and their subsequent disappearance paralleled a decrease in overall AChR levels. Quantitative autoradiography showed a reduction of over 75% in the density of AChR on the surface of well differentiated, cross-striated myotubes. Thus the appearance of AChR on the cell surface, its condensation into clusters, and finally its depletion seem to be sequential events in the differentiation of skeletal muscle in culture in the absence of direct neuronal influence.
ESTHER : Prives_1976_Cell_7_543
PubMedSearch : Prives_1976_Cell_7_543
PubMedID: 954086

Title : Detection of hydroxyproline in preparations of acetylcholinesterase from the electric organ of the electric eel -
Author(s) : Anglister L , Rogozinski S , Silman I
Ref : FEBS Letters , 69 :129 , 1976
PubMedID: 992021

Title : Proceedings: A water-soluble acetylcholine receptor fraction from electric organ tissue of the electric eel Electrophorus electricus -
Author(s) : Kalderon N , Silman I
Ref : Isr J Med Sci , 11 :1192 , 1975
PubMedID: 1205776

Title : Proceedings: Appearance and disappearance of acetylcholine receptor during differentiation of embryonic chick skeletal muscle in vitro -
Author(s) : Prives J , Silman I , Amsterdam A
Ref : Isr J Med Sci , 11 :1192 , 1975
PubMedID: 1205775

Title : Molecular structure and catalytic activity of membrane-bound acetylcholinesterase from Electric Organ Tissue of the Electric Eel -
Author(s) : Silman I , Dudai Y
Ref : Croatica Chemica Acta , 47 :181 , 1975
PubMedID:

Title : Proceedings: Animal model for myasthenia gravis: acetylcholine receptor-induced myasthenia in rabbits, guinea pigs and monkeys -
Author(s) : Tarrab-Hazdai R , Aharonov A , Abramsky O , Silman I , Fuchs S
Ref : Isr J Med Sci , 11 :1390 , 1975
PubMedID: 815200

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 : Proceedings: Thymus, acetylcholine receptor and myasthenia gravis -
Author(s) : Aharonov A , Tarrab-Hazdai R , Abramsky O , Silman I , Fuchs S
Ref : Isr J Med Sci , 11 :1390 , 1975
PubMedID: 1218989

Title : Affinity chromatography of acetylcholinesterase -
Author(s) : Dudai Y , Silman I
Ref : Methods Enzymol , 34 :571 , 1974
PubMedID: 4449474

Title : The effects of solubilization procedures on the release and molecular state of acetylcholinesterase from electric organ tissue -
Author(s) : Dudai Y , Silman I
Ref : Journal of Neurochemistry , 23 :1177 , 1974
PubMedID: 4375706

Title : The molecular weight and subunit structure of acetylcholinesterase preparations from the electric organ of the electric eel -
Author(s) : Dudai Y , Silman I
Ref : Biochemical & Biophysical Research Communications , 59 :117 , 1974
PubMedID: 4842295

Title : Immunochemical and enzymic properties of the electric eel acetylcholinesterase-antiacetylcholinesterase system -
Author(s) : Gurari D , Silman I , Fuchs S
Ref : European Journal of Biochemistry , 43 :179 , 1974
PubMedID: 4134781

Title : Chemical modification of electric eel acetylcholinesterase by tetranitromethane -
Author(s) : Fuchs S , Gurari D , Silman I
Ref : Archives of Biochemistry & Biophysics , 165 :90 , 1974
PubMedID: 4441088

Title : Spectral evidence for the presence of tryptophan in the binding site of acetylcholinesterase -
Author(s) : Shinitzky M , Dudai Y , Silman I
Ref : FEBS Letters , 30 :125 , 1973
PubMedID: 11947075

Title : The effect of Ca(2+) on interaction of acetylcholinesterase with subcellular fractions of electric organ tissue from the electric eel -
Author(s) : Dudai Y , Silman I
Ref : FEBS Letters , 30 :49 , 1973
PubMedID: 11947059

Title : Effect of cholinesterase inhibitors on active potassium influx in monkey erythrocytes -
Author(s) : Giberman E , Silman I , Edery H
Ref : Biochemical Pharmacology , 22 :271 , 1973
PubMedID: 4202811

Title : Molecular structures of acetylcholinesterase from electric organ tissue of the electric eel - Dudai_1973_Proc.Natl.Acad.Sci.U.S.A_70_2473
Author(s) : Dudai Y , Herzberg M , Silman I
Ref : Proceedings of the National Academy of Sciences of the United States of America , 70 :2473 , 1973
Abstract : Three purified molecular forms of acetylcholinesterase (EC 3.1.1.7) with sedimentation coefficients of 18 S, 14 S, and 11 S were studied by analytical ultracentrifugation and electron microscopy. The three species have molecular weights of (1.1 +/- 0.1) x 10(6), (7.5 +/- 1.5) x 10(5), and (3.35 +/- 0.25) x 10(5), respectively. Electron micrographs reveal that the 18S and 14S forms are asymmetric, composed of a head, containing a large number of subunits, and an elongated tail. The 11S form of acetylcholinesterase is apparently a tetrameric structure devoid of the tail. Maleylation of 18S and 14S acetylcholinesterases abolishes their tendency to aggregate at low ionic strength.
ESTHER : Dudai_1973_Proc.Natl.Acad.Sci.U.S.A_70_2473
PubMedSearch : Dudai_1973_Proc.Natl.Acad.Sci.U.S.A_70_2473
PubMedID: 4517659

Title : Purification by affinity chromatography of acetylcholinesterase from electric organ tissue of the electric eel subsequent to tryptic treatment -
Author(s) : Dudai Y , Silman I , Kalderon N , Blumberg S
Ref : Biochimica & Biophysica Acta , 268 :138 , 1972
PubMedID: 5018273

Title : Purification by affinity chromatography of the molecular forms of acetylcholinesterase present in fresh electric-organ tissue of electric eel - Dudai_1972_Proc.Natl.Acad.Sci.U.S.A_69_2400
Author(s) : Dudai Y , Silman I , Shinitzky M , Blumberg S
Ref : Proceedings of the National Academy of Sciences of the United States of America , 69 :2400 , 1972
Abstract : An acetylcholinesterase inhibitor-Sepharose conjugate was prepared by coupling a derivative of the powerful acetylcholinesterase inhibitor, N-methylacridinium, to CNBr-activated Sepharose. Use of this conjugate permitted direct purification, by affinity chromatography, of the two molecular forms of acetylcholinesterase, 14 and 18 S, present in fresh electric organ tissue. The purified 14S and 18S acetylcholinesterases retained the capacity to aggregate at low ionic strength displayed by crude extracts of the enzyme. The major polypeptide components of the 14S and 18S enzymes, as revealed by acrylamide gel electrophoresis, closely resemble those observed in the 11S form of acetylcholinesterase, previously purified after tryptic digestion of electric-organ tissue.
ESTHER : Dudai_1972_Proc.Natl.Acad.Sci.U.S.A_69_2400
PubMedSearch : Dudai_1972_Proc.Natl.Acad.Sci.U.S.A_69_2400
PubMedID: 4506759

Title : The subunits of an acetylcholinesterase preparation purified from trypsin-treated electric eel tissue -
Author(s) : Dudai Y , Silman I
Ref : FEBS Letters , 16 :324 , 1971
PubMedID: 11945972

Title : A method for the purification of acetylcholinesterase by affinity chromatography -
Author(s) : Kalderon N , Silman I , Blumberg S , Dudai Y
Ref : Biochimica & Biophysica Acta , 207 :560 , 1970
PubMedID: 5452681

Title : Acetylcholine receptor: covalent attachment of depolarizing groups at the active site - Silman_1969_Science_164_1420
Author(s) : Silman I , Karlin A
Ref : Science , 164 :1420 , 1969
Abstract : Following reduction of the acetylcholine receptor in the electroplax with dithiothreitol, the quaternary ammonium compounds bromoacetylcholine bromide and the p-nitrophenyl ester of (p-carboxyphenyl) trimethylammonium iodide react near the active site probably with a sulfhydryl group. The covalently attached quaternary ammonium moieties additionally interact with the active site noncovalently to activate the receptor and cause depolarization of the cell.
ESTHER : Silman_1969_Science_164_1420
PubMedSearch : Silman_1969_Science_164_1420
PubMedID: 5783718

Title : Microenvironmental effects on enzyme activity -
Author(s) : Silman I
Ref : Journal of General Physiology , 54 :50 , 1969
PubMedID: 19873655

Title : Papain-collodion membranes. I. Preparation and properties -
Author(s) : Goldman R , Kedem O , Silman I , Caplan SR , Katchalski E
Ref : Biochemistry , 7 :486 , 1968
PubMedID: 5642388

Title : Effect of local pH change caused by substrate hydrolysis on the activity of membrane-bound acetylcholinesterase -
Author(s) : Silman I , Karlin A
Ref : Proceedings of the National Academy of Sciences of the United States of America , 58 :1664 , 1967
PubMedID:

Title : Water-insoluble derivatives of enzymes, antigens, and antibodies -
Author(s) : Silman I , Katchalski E
Ref : Annual Review of Biochemistry , 35 :873 , 1966
PubMedID: 5329475

Title : Some water-insoluble papain derivatives -
Author(s) : Silman I , Albu-Weissenberg M , Katchalski E
Ref : Biopolymers , 4 :441 , 1966
PubMedID: 5948638