Active Site Gorge Of Acetylcholinesterase Research Articles

Binding interactions and in silico ADME prediction of isoconessimine derivatives as potent acetylcholinesterase inhibitors

In pursuit of new acetylcholinesterase (AChE) inhibitors for treating Alzheimer's disease (AD), a series of ten previously synthesized isoconessimine compounds (7a-7j) was in silico investigated for their binding interactions with AChE and pharmacokinetics based on absorption, distribution, metabolism, and excretion (ADME) properties using molecular docking, ONIOM (Our own N-layered Integrated molecular Orbital and molecular Mechanics) method and SwissADME tools. Docking experiments showed that all compounds bind within the active site gorge of AChE (PDB entry 1C2B), posing its aryloxy-substitutional ethyl group to catalytic site and conessine skeleton to peripheral anionic site. ONIOM interaction energy was used as an ONIOM score to improve docking score, and it ranked 7b as the most potent AChE inhibitor, in agreement with previous experiment. Residues, ASP74, TRP86, GLY122, GLU202, TRP286, GLU292, SER293, ILE294, TYR337, TYR341, and HIS447 were identified as important for the binding of the AChE-isoconessimine complex. The SwissADME investigation suggested that four compounds (7a, 7c, 7d and 7f) agree with the rules of drug-likeness. The steric and electronic effects on the aryloxy-substitutional ethyl group as important factors in the AChE inhibition were also discussed, which brings a better understanding of Alzheimer's disease drug development.

Bioactivity and molecular docking studies of selected plant compounds.

The shortcomings of synthetic pesticides are becoming increasingly evident. In the pursuit of looking for alternative, environmentally friendly pesticides, the potential use of two naturally occurring sesquiterpenes, α-bisabolol and bisabolol oxide A, as bactericides or acaricides, was investigated against three gram-positive and three gram-negative bacteria, and the eggs and larvae of the cattle tick Hyalomma scupense, respectively. The primary targeted site of synthetic insecticides is acetylcholinesterase (AChE), so the inhibitory activity of both sesquiterpenes was evaluated. Then, molecular docking and molecular mechanics generalized Born surface area (MM-GBSA)studies were performed to gain insight into their binding mode in the AChE active site. Bisabolol oxide A showed good activity against Staphylococcus aureus, Escherichia coli, and Salmonella enteritidis with inhibition zones ranging from 16 to 34 mm and minimal inhibitory concentration (CIM) of 3.125 mg mL-1. Ovicidal and larvicidal assays revealed promising activity of α-bisabolol and bisabolol oxide A against H. scupense, with bisabolol oxide A being more effective against larval ticks with lethal concentration (LC50) value of 0.78%. Both compounds also displayed potent anti-AChE activity with inhibition concentration (IC50) values of 37.09 and 28.14 μg mL-1, respectively. Furthermore, α-bisabolol and bisabolol oxide A exhibited good and comparable docking scores (-7.289 and -7.139 Kcal mol-1, respectively) and were found to accommodate in the active-site gorge of AChE via hydrogen bonding and hydrophobic interactions. Bisabolol oxide A and, to a lesser degree, α-bisabolol are active against bacteria and ectoparasites and may represent an economical and sustainable alternative to toxic synthetic pesticides to control pathogens. © 2024 Society of Chemical Industry.

In vitro and in silico analysis of novel astaxanthin-s-allyl cysteine as an inhibitor of butyrylcholinesterase and various globular forms of acetylcholinesterases

In Alzheimer's disease (AD) and diabetes-associated cognitive decline, the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity is increased. AChE exists as different globular molecular forms: tetramer (G4), dimer (G2) and monomer (G1). In adult brain, G4 form is abundant however in AD, the ratio of lower molecular forms (G1) to G4 form increased. Hence, the present study delineated the inhibition of novel astaxanthin-s-allyl cysteine (AST-SAC) against BChE and various molecular forms of AChE. Cobra venom, human erythrocyte and Electrophorus electricus was used as source of G1, G2 and G4 form of AChE. AST-SAC showed inhibition against G1 (IC50 = 0.72 μM, competitive, Ki = 0.66 μM), G2 (IC50 = 0.65 μM, mixed, Ki = 0.50 μM) and G4 (IC50 = 0.67 μM, competitive, Ki = 0.67 μM) form of AChE. AST-SAC inhibited human brain AChE (IC50 = 0.84 μM, competitive, Ki = 0.53 μM) and human serum BChE (IC50 = 0.80 μM, competitive, Ki = 0.58 μM). In silico analysis revealed the interaction of AST-SAC with the amino acids present in peripheral anionic and catalytic site of human AChE and BChE. Molecular dynamics simulation confirmed the stable interaction of AST-SAC in the active site gorge of AChE and BChE.

Potent Acetylcholinesterase Selective and Reversible Homodimeric Agent Based on Tacrine for Theranostics.

Acetylcholinesterase (AChE) has been an important biomarker for diagnosing Alzheimer's disease (AD), due to reduction in AChE activity in post-mortem brains of AD patients. A potent, selective, and reversible homodimeric inhibitor of AChE, 5-amino- N1, N3-bis(2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethyl)isophthalamide (compound 4), was synthesized by using 9-alkyl(1,2,3,4-tetrahydroacridine) pharmacophore with appended functionality. In the present work, we report the synthesis of this bivalent inhibitor of AChE. The homodimeric ligand structure was designed and studied with molecular docking tools, which revealed its high affinity and interactions with active site gorge of AChE, which includes both catalytic active site (CAS) and peripheral active site (PAS). The IC50 value of this bivalent inhibitor for AChE and BuChE were 0.54 ± 0.06 and 32.49 ± 1.2 nM, respectively, with a selectivity ratio of 60.16 toward AChE. The designed ligand also showed potent inhibitory properties on PAS activity as well as on AChE-induced amyloid aggregation with low cytotoxicity on rat hippocampal neurons. The AFM images further corroborated the Aβ1-42 aggregation inhibition by compound 4 to an extent similar to bis(7)-tacrine. Moreover, the bivalent ligand was also proven to be of neurogenic potential due to its ability to induce S-phase post-treatment in rat hippocampal neuronal cells. On the basis of initial results, the agent could be further explored for its theranostic value clinically, which gives the possibility of tracing the AChE levels by molecular imaging techniques in correlation with progression of neurocognitive disorders like AD for better therapy response and patient management.

The inhibition of acetylcholinesterase by a brain-targeting polylysine-ApoE peptide: biochemical and structural characterizations.

The in-trans delivery of protein therapeutics across the blood-brain barrier by K16ApoE peptide carrier has been demonstrated to improve the neurological symptoms and increase the life-span of late-infantile neuronal ceroid lipofuscinosis (LINCL) mice. However, acute toxicity of K16ApoE was observed in LINCL mice resulting in a narrow therapeutic index, limiting the potential of translating the K16ApoE into a viable drug delivery system. This study aims to unravel the toxic mechanism of action. We hypothesized that the toxic response towards the peptide was induced by inhibition of acetylcholinesterase (AChE) activity at neuro-muscular junction. Here, results from the dose-response study suggested that AChE activity was inhibited by K16ApoE at either low or high doses but not at the mid-dose where a significant increase in AChE activity was observed. Meanwhile, molecular docking simulations showed that the N-terminus of K16ApoE is capable of binding to the active site gorge of AChE. In addition to a favorable spatial orientation, this docking pose also revealed strong surface charge interactions which may account for the observed inhibitory effect. While statistical analysis of the dose response and survival ratio suggested that AChE is not the primary mechanism of action for the acute toxicity of K16ApoE, both biochemical evidence and structural analysis have assigned indirect but critical roles for AChE in the overall toxicity mechanism of this peptide carrier.

Dynamic structure based pharmacophore modeling of the Acetylcholinesterase reveals several potential inhibitors

Acetylcholinesterase is a critical enzyme that regulates neurotransmission by catalyzing the breakdown of neurotransmitter acetylcholine in synapses of the nervous system. It is an important target for therapeutic drugs that treat Alzheimer’s disease. Since, the degree of flexibility of the side chains of the residues in the active-site gorge of Acetylcholinesterase is diverse it results in different bound ligand conformations. The side-chain conformations of Ser293, Tyr341, Leu76, and Val73 are flexible, while the side-chain conformations of Tyr72, Tyr 124, Ser125, Phe295, and Arg296 appear to be fixed. In this study, multi-conformation dynamic pharmacophore models from the donepezyl-binding pocket based on highly populated structures chosen from molecular dynamics simulations were used for screening compounds that can properly bind acetylcholinesterase. Based on these structures, three pharmacophore models were generated. Consequently, 14 hits were retrieved as final candidates by utilizing virtual screening of ZINC database and molecular docking.

Application of a dynamic in vitro model with real-time determination of acetylcholinesterase activity for the investigation of tabun analogues and oximes

Tabun-inhibited acetylcholinesterase (AChE) is rather resistant towards reactivation by oximes in vitro while in vivo experiments showed some protection of animals poisoned by this chemical warfare nerve agent after treatment with an oxime and atropine. In addition, AChE inhibited by close tabun analogues, N,N-diethyltabun and N,N-di-n-propyltabun was completely resistant towards reactivation by oximes. In order to get more insight into potential mechanisms of this oxime resistance experiments with these toxic agents and the oximes obidoxime, 2-PAM, MMB-4 and HI-6 were performed utilizing a dynamic model with real-time determination of AChE activity. This experimental setup allowed the investigation of reactivation with minimized side reactions. The determined reactivation constants with tabun-inhibited human AChE were in good agreement with previously reported constants determined with a static model. N,N-diethyl- and N,N-di-n-propyltabun-inhibited human AChE could not be reactivated by oximes which indicates that the inadequate oxime effect was not due to re-inhibition by phosphonyloximes. Additional experiments with tabun-inhibited human and Rhesus monkey AChE revealed that no reactivation occurred with HI-6. These data give further support to the assumption that an interaction of tabun with residues in the active site gorge of AChE prevents effective reactivation by oximes, a mechanism which may also be the reason for the total oxime resistance of N,N-diethyl- and N,N-di-n-propyltabun-inhibited human AChE.

Cholinesterase inhibitory activity versus aromatic core multiplicity: A facile green synthesis and molecular docking study of novel piperidone embedded thiazolopyrimidines

Novel thiazolopyrimidine derivatives have been synthesized via microwave assisted, domino cascade methodology in ionic liquid and evaluated in vitro for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Among the newly synthesized compounds 6d, 6a, 6e and 6b displayed higher AChE inhibitory activity than standard drug, galanthamine, with IC50 values of 0.53, 1.47, 1.62 and 2.05μM, respectively. Interestingly, all the compounds except for 6m–r and 6x displayed higher BChE inhibitory potentials than galanthamine with IC50 values ranging from 1.09 to 18.56μM. Molecular docking simulations for 6d possessing the most potent AChE and BChE inhibitory activities, disclosed its binding interactions at the active site gorge of AChE and BChE enzymes.

Flexibility of active-site gorge aromatic residues and non-gorge aromatic residues in acetylcholinesterase

AbstractThe presence of an unusually large number of aromatic residues in the active site gorge of acetylcholinesterase is a subject of great interest. Flexibility of these residues has been suspected to be a key player in controlling the ligand traversal in the gorge. This raises the question of whether the over-representation of aromatic residues in the gorge implies higher-than-normal flexibility of these residues. The current study suggests that it does not. Large changes in the hydrophobic cross-sectional area due to dihedral oscillations are probably the reason of their presence in the gorge.

Energetics of Ortho-7 (oxime drug) translocation through the active-site gorge of tabun conjugated acetylcholinesterase.

Oxime drugs translocate through the 20 Å active-site gorge of acetylcholinesterase in order to liberate the enzyme from organophosphorus compounds’ (such as tabun) conjugation. Here we report bidirectional steered molecular dynamics simulations of oxime drug (Ortho-7) translocation through the gorge of tabun intoxicated enzyme, in which time dependent external forces accelerate the translocation event. The simulations reveal the participation of drug-enzyme hydrogen bonding, hydrophobic interactions and water bridges between them. Employing nonequilibrium theorems that recovers the free energy from irreversible work done, we reconstruct potential of mean force along the translocation pathway such that the desired quantity represents an unperturbed system. The potential locates the binding sites and barriers for the drug to translocate inside the gorge. Configurational entropic contribution of the protein-drug binding entity and the role of solvent translational mobility in the binding energetics is further assessed.

Effect of electromagnetic field on acetylcholinesterase activity: In vitro study

The in vitro effects of extremely low frequency (ELF)-electromagnetic field (EMF) (frequencies of 50, 100, 217 Hz, induction 300 µT) on the structure and activity of acetylcholinesterase (AChE) was investigated. AChE activity was measured with UV-vis spectroscopy. Circular dichroism spectroscopy and fluorescence spectroscopy were used for investigating the secondary and tertiary structure of enzyme. The results demonstrated that the ELF-EMF decreased enzyme activity and the main inactivation took place in 217 Hz (p<0.05). It can be concluded that increasing the frequencies of ELF-EMF caused an increase in the helicity of AChE; therefore, under ELF-EMF, the α-helix structure can induce the secondary structure of AChE. Also, the intrinsic fluorescence decreased by increasing the frequency of field. In conclusion, according to changes observed in the secondary and tertiary structure of enzyme, it is proposed that these fields are able to affect the structure and dynamics of the active site gorge of AChE. Key words: Acetylcholinestrase, circular dichroism spectroscopy, extremely low frequency electromagnetic field, fluorescence spectroscopy, UV-visible spectroscopy.

New Huprine Derivatives Functionalized at Position 9 as Highly Potent Acetylcholinesterase Inhibitors

A series of 24 huprine derivatives diversely functionalized at position 9 have been synthesized and evaluated for their inhibitory activity against human recombinant acetylcholinesterase (AChE). These derivatives were prepared in one to five steps from huprine 1 bearing an ester function at position 9. Ten analogues (1, 2, 6-9, 13-15, and 23) are active in the low nanomolar range (IC(50) <5 nM), very close to the parent compound huprine X. Compounds 2, 6, and 7 show a very good selectivity for AChE, with AChE inhibitory activities 700-1160-fold higher than those for butyrylcholinesterase (BChE). The inhibitory potency of these compounds decreases with the steric bulk of the substituents at position 9. According to docking simulations, small substituents fit into the acyl-binding pocket, whereas the larger ones stick out of the active site gorge of AChE. Determination of the kinetic parameters of three of the most potent huprines (2, 6, and 7) showed that most of the difference in K(D) is accounted by a decrease in k(on) , which is correlated to the increase of the substituent size. A first in vivo evaluation has been performed in mice for the most active compound 2 (IC(50) =1.1 nM) and showed a rather weak toxicity (LD(50) =40 mg kg(-1) ) and an ability to cross the blood-brain barrier with doses above 15 mg kg(-1).

Targeted Oxidation of Torpedo californica Acetylcholinesterase by Singlet Oxygen

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.

Long Route or Shortcut? A Molecular Dynamics Study of Traffic of Thiocholine within the Active-Site Gorge of Acetylcholinesterase

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.

An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of α/β-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005–3.0 mM, with 317 pM human butyrylcholinesterase in vitro, resulted in rates of production of thiocholine that were accurately described by simple Michaelis–Menten kinetics, with a K m of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis–Menten kinetics, with a k i of 3048 nM −1 h −1, and a K D of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k i increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 and 0.3 nM gave k is of 1.2 and 19.3 nM −1 h −1, respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.

Bivalent Ligands Derived from Huperzine A as Acetylcholinesterase Inhibitors

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-à-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.

Complexes of Alkylene-Linked Tacrine Dimers with Torpedo californica Acetylcholinesterase: Binding of Bis(5)-tacrine Produces a Dramatic Rearrangement in the Active-Site Gorge

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.

Crystal structures of acetylcholinesterase in complex with HI-6, Ortho-7 and obidoxime: Structural basis for differences in the ability to reactivate tabun conjugates

Inhibition of acetylcholinesterase (AChE) by organophosphorus compounds (OPs) such as pesticides and nerve agents causes acute toxicity or death of the intoxicated individual. The inhibited AChE may be reactivated by certain oximes as antidotes for clinical treatment of OP-intoxications. Crystal structures of the oximes HI-6, Ortho-7 and obidoxime in complex with Mus musculus acetylcholinesterase (mAChE) reveal different roles of the peripheral anionic site (PAS) in the binding of the oximes. A limited structural change of the side chains of Trp286 and Asp74 facilitates the intercalation of the 4-carboxylamide pyridinium ring of HI-6 between the side chains of Tyr124 and Trp286. The 2-carboxyimino pyridinium ring of HI-6 is accommodated at the entrance of the catalytic site with the oximate forming a hydrogen bond to the main-chain nitrogen atom of Phe295. In contrast to HI-6, the coordination of Ortho-7 and obidoxime within the PAS is facilitated by an extended structural change of Trp286 that allows one of the carboxyimino pyridinium rings to form a cation-π interaction with the aromatic groups of Tyr72 and Trp286. The central chain of Ortho-7 and obidoxime is loosely coordinated in the active-site gorge, whereas the second carboxyimino pyridinium ring is accommodated in the vicinity of the phenol ring of Tyr337. The structural data clearly show analogous coordination of Ortho-7 and obidoxime within the active-site gorge of AChE. Different ability to reactivate AChE inhibited by tabun is shown in end-point reactivation experiments where HI-6, Ortho-7 and obidoxime showed an efficiency of 1, 45 and 38%, respectively. The low efficiency of HI-6 and the significantly higher efficiency of Ortho-7 and obidoxime may be explained by the differential binding of the oximes in the PAS and active-site gorge of AChE.

(21) Measuring carbamoylation and decarbamoylation rate constants by continuous assay of AChE

The active site gorge of acetylcholinesterase (AChE) contains two distinct sites of ligand interaction: an acylation site (A-site) at the base of the gorge and a peripheral site (P-site) near the mouth of the gorge at the enzyme surface. While the role of the P-site in catalysis has been elusive, our recent studies have shown that it serves as an intermediate binding site for cationic ligands as they proceed to the A-site. P-site binding also can lead to allosteric activation of the acylation step and/or to steric blockade, defined as an equal reduction in the association and dissociation rate constants for the binding of a second ligand to the A-site when the P-site is occupied. Compelling evidence that allosteric activation occurs at the acylation step came from examination of the hydrolysis of a close substrate analog of acetylcholine, the acetanilide ATMA [J.L. Johnson, B. Cusack, M.P. Davies, A. Fauq, T.L. Rosenberry, Biochemistry 42 (2003) 5438–5452]. The acylation step in the AChE hydrolysis of ATMA can be isolated because of the slow cleavage of the anilide bond, revealing the positive deviation from Michealis-Menten kinetics that defines substrate activation. However, not all substrates that bind to the P-site show allosteric activation of acylation. In fact, the only substrates that we have identified thus far are ATMA and, to a lesser extent, acetylthiocholine. Carbamates are also poor substrates of AChE, but they differ from ATMA in that both the formation and hydrolysis of the intermediate acyl enzyme are slow. Thus, the hydrolysis of carbamates by AChE can be resolved into two distinct catalytic steps, carbamoylation and decarbamoylation. The ability to kinetically isolate these two catalytic steps also allows us to investigate which step, if any, is subject to allosteric activation.

(37) Ligand-induced conformational changes in residues flanking the active site gorge of acetylcholinesterase

(37) Ligand-induced conformational changes in residues flanking the active site gorge of acetylcholinesterase