The ‘aromatic patch’ of three proximal residues in the human acetylcholinesterase active centre allows for versatile interaction modes with inhibitors

Ariel, Naomi; Ordentlich, Arie; Barak, Dov; Bino, Tamar; Velan, Baruch; Shafferman, Avigdor

doi:10.1042/bj3350095

Cited by 93 publications

(96 citation statements)

References 27 publications

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“…The decrease of the W86A HuAChE reactivity toward the VX S and VX R enantiomers as compared to the wild type enzyme (the values of bimolecular rate constants decreased by 4500-fold and almost 1000-fold respectively, see Table 2) was compatible with what was observed in the past for other covalent and noncovalent charged ligands [15,24,30]. The corresponding effects of the replacement of Trp86 by phenylalanine were much smaller and consistent with the well-established role of the aromatic residue at position-86 as the main interaction locus of the charged moieties of AChE ligands [30,31].…”

Section: Stereoselectivity Precipitated By Charge Interactions With Tsupporting

confidence: 70%

Flexibility versus “rigidity” of the functional architecture of AChE active center

Shafferman

Barak

Stein

et al. 2008

Chemico-Biological Interactions

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

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Section: Stereoselectivity Precipitated By Charge Interactions With Tsupporting

confidence: 70%

Flexibility versus “rigidity” of the functional architecture of AChE active center

Shafferman

Barak

Stein

et al. 2008

Chemico-Biological Interactions

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“…(R p )-DMBMP-AChE yields an emission maximum of 459 nm, a difference of 51 nm from the (S p )-DMBMPAChE (Table III). directed toward the oxyanion hole, and the alkyl carbamoyl group pointing toward the acyl pocket (25,31,32). Similar to the tetrahedral phosphoryl and phosphonyl conjugates, the trigonal carbamoylated enzymes produce very little perturbation at positions 76, 262, and 287 (Table IV).…”

Section: Fig 3 Fluorescence Emission Spectra Of Acrylodan-labeled Ementioning

confidence: 85%

“…This applies to the dimethylphosphoryl conjugate, all of the S p methylphosphonyl conjugates and perhaps to the methylcarbamoyl conjugate. It should be noted that all of these compounds would allow for insertion of the phosphoryl and carbamoyl oxygen in the oxyanion hole formed by hydrogen bonds donors from amide backbone hydrogens at Gly 121 , Gly 122 , and Ala 204 without deforming the acyl pocket (31). By contrast, the diethyl and diisopropyl phosphoryl conjugates and the corresponding R p phosphonyl-AChE derivatives cannot stabilize their phosphoryl or phosphonyl oxygen in the oxyanion hole unless the alkoxy moiety perturbs or moves out of the acyl pocket.…”

Section: Fig 3 Fluorescence Emission Spectra Of Acrylodan-labeled Ementioning

confidence: 99%

Inhibitors of Different Structure Induce Distinguishing Conformations in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase

Shi

Radić

Taylor

2002

Journal of Biological Chemistry

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We have shown previously that association of reversible active site ligands induces a conformational change in an omega loop (⍀ loop), Cys 69 -Cys 96 , of acetylcholinesterase. The fluorophore acrylodan, site-specifically incorporated at positions 76, 81, and 84, on the external portion of the loop not lining the active site gorge, shows changes in its fluorescence spectrum that reflect the fluorescent side chain moving from a hydrophobic environment to become more solvent-exposed. This appears to result from a movement of the ⍀ loop accompanying ligand binding. We show here that the loop is indeed flexible and responds to conformational changes induced by both active center and peripheral site inhibitors (gallamine and fasciculin). Moreover, phosphorylation and carbamoylation of the active center serine shows distinctive changes in acrylodan fluorescence spectra at the ⍀ loop sites, depending on the chirality and steric dimensions of the covalently conjugated ligand. Capping of the gorge with fasciculin, although it does not displace the bound ligand, dominates in inducing a conformational change in the loop. Hence, the ligand-induced conformational changes are distinctive and suggest multiple loop conformations accompany conjugation at the active center serine. The fluorescence changes induced by the modified enzyme may prove useful in the detection of organophosphates or exposure to cholinesterase inhibitors. Acetylcholinesterase (AChE)1 plays a pivotal role in neurotransmission by terminating the action of neurotransmitter, acetylcholine, at neuromuscular junction and other cholinergic synapses (1-3). AChE is one of most efficient enzymes known with hydrolysis of its natural substrate reaching diffusion-controlled limits. Inhibitors of AChE target two sites in the active site gorge: an active center at the base of a narrow gorge 20 Å in depth and a peripheral site at the gorge rim (4). At the active center, a residue triad (Ser 203 -Glu 334 -His 447 ) promotes acyl transfer and hydrolysis of the substrate, whereas Trp 86 at the gorge base primarily stabilizes choline moiety of the substrate through a cationinteraction. Active site inhibitors block substrate binding either by associating with the tryptophan in the choline binding site (tacrine and edrophonium) or by reacting irreversibly with catalytic serine (carbamates and organophosphates). Peripheral site inhibitors, such as propidium and gallamine, inhibit catalytic activity through both steric blockade and allosterically altering catalytic efficiency of the active center residues (4 -8).To elucidate the conformational changes associated with mechanistically distinctive inhibitors, we developed a means for physically monitoring the conformation of purified mouse AChE by site-directed labeling with an environmentally sensitive fluorophore, acrylodan. Six single cysteine mutants were prepared for acrylodan conjugation (Fig. 1). Three were on the Cys 69 -Cys 96 omega loop (⍀ loop) flanking the active site gorge: L76C near the tip of the loop, and E81C and E...

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“…and Ariel et al 30 For example, 84% of the aminoacids of the active site of HuAChE were identical to the residues of the TcAChE active site as shown in Table 2.…”

mentioning

confidence: 97%

“…The identification of the residues belonging to the active site of HuAChE was obtained by the sequence alignment between HuAChE and the crystallographic structure of AChE from Torpedo californica (TcAChE, PDB code 1EA5) 27 and also by comparison with the residues reported by Shafferman et al, 28 Enyedy et al 29 and Ariel et al 30 For example, 84% of the aminoacids of the active site of HuAChE were identical to the residues of the TcAChE active site as shown in Table 2.…”

Section: Homology Modeling and Active Site Determinationmentioning

confidence: 99%

Molecular dynamics of the interaction of pralidoxime and deazapralidoxime with acetylcholinesterase inhibited by the neurotoxic agent tabun

Gonçalves¹,

França²,

Wilter³

et al. 2006

J. Braz. Chem. Soc.

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Reativadores eficientes de Aceticolinesterase são fundamentais para o desenvolvimento de antídotos contra o envenenamento por pesticidas neurotóxicos e agentes de guerra química. Todavia, o mecanismo da reação de reativação e as características estruturais dos reativadores conhecidos são pouco compreendidos. Com o objetivo de estudar o comportamento dinâmico e o efeito da carga líquida do antídoto na reativação desta enzima, foi conduzido um estudo por dinâmica molecular da acetilcolinesterase humana inibida por tabun em complexo com o antídoto pralidoxima e com seu análogo deazapralidoxima nas formas neutra e aniônica. Os resultados mostraram que a carga positiva da pralidoxima é importrante para sua admissão e permanência dentro do sítio ativo. Além disso, os análogos, diferente da pralidoxima, quando colocados dentro do sítio ativo, se distanciam do resíduo serina fosforilado da enzima e são repelidos pelo potencial eletrostático na entrada do canal que conduz ao sítio ativo.Efficient acetylcholinesterase reactivators are fundamental for the development of antidotes against poisoning by neurotoxic pesticides and chemical warfare agents. However, the mechanism of the reactivation reaction and the structural characteristics of the known reactivators are poorly understood. In order to study the dynamic behavior and the effect of the antidote net charge in the reactivation of this enzyme, we carried out a molecular dynamics study of human acetylcholinesterase inhibited by tabun in complex with the antidote pralidoxime and with its deaza analogues in the neutral and anionic forms. Results show that the positive charge of pralidoxime is important for its admission and permanence inside the active site. Also, the analogues, unlike pralidoxime, when forced inside the active site, move away from the phosphorilated serine residue of the enzyme and are repelled by the electrostatic potential at the entrance of the channel that conducts to the active site. Keywords: acetylcholinesterase, molecular dynamics, tabun, antidotes, neurotoxic agents IntroductionThe intensive use of neurotoxic organophosphorous compounds as pesticides in agriculture, as well as their potential use as mass destruction agents in chemical warfare, has attracted attention to the development of efficient antidotes for this type of poisoning.1,2 However, the knowledge on the appropriate treatment for patients exposed to this kind of compounds is limited to few groups of physicians around the world. 1,2One particularly important family of lethal tactical warfare chemicals is the group known as the nerve agents, which are closely related in chemical structure and biological action to many commonly used organophosphorous insecticides, but which are much more lethal. 1,2The nerve agents are esters of phosphoric acid and are potent inhibitors of acetylcholinesterase, a fundamental enzyme for ending nervous impulses. These compounds inhibit all acetylcholinesterases, including the human enzyme (HuAChE), by phosphorylating a serine hydroxyl group (Ser203 in ...

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The ‘aromatic patch’ of three proximal residues in the human acetylcholinesterase active centre allows for versatile interaction modes with inhibitors

Cited by 93 publications

References 27 publications

Flexibility versus “rigidity” of the functional architecture of AChE active center

Flexibility versus “rigidity” of the functional architecture of AChE active center

Inhibitors of Different Structure Induce Distinguishing Conformations in the Omega Loop, Cys69–Cys96, of Mouse Acetylcholinesterase

Molecular dynamics of the interaction of pralidoxime and deazapralidoxime with acetylcholinesterase inhibited by the neurotoxic agent tabun

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