oxime interaction reactivation of tabun-inhibited human AChE. Unable to facilitate HI-6-mediated reactivation of tabun-hAChE. Induced a 2-2.5-fold enhancement of the bimolecular rate constant for K027 and HLo-7 Artursson_2009_Toxicology_265_108
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p.Y337F Tyr337Phe (p.Y368F Tyr368Phe in primary sequence with 31 amino-acids signal peptide) Signal transduction, very low effect on catalysis and inhibition; Substrate inhibition identical to wild type
The nerve agent tabun inhibits the essential enzyme acetylcholinesterase (AChE) by a rapid phosphoramidation of the catalytic serine residue. Oximes, such as K027 and HLo-7, can reactivate tabun-inhibited human AChE (tabun-hAChE) whereas the activity of their close structural analogue HI-6 is notably low. To investigate HI-6, K027 and HLo-7, residues lining the active-site gorge of hAChE were substituted and the effects on kinetic parameters for reactivation were determined. None of the mutants (Asp74Asn, Asp74Glu, Tyr124Phe, Tyr337Ala, Tyr337Phe, Phe338Val and Tyr341Ala) were able to facilitate HI-6-mediated reactivation of tabun-hAChE. In contrast, Tyr124Phe and Tyr337Phe induce a 2-2.5-fold enhancement of the bimolecular rate constant for K027 and HLo-7. The largest effects on the dissociation constant (3.5-fold increase) and rate constant (20-fold decrease) were observed for Tyr341Ala and Asp74Asn, respectively. These findings demonstrate the importance of residues located distant from the conjugate during the reactivation of tabun-hAChE.
Title: The 'aromatic patch' of three proximal residues in the human acetylcholinesterase active centre allows for versatile interaction modes with inhibitors Ariel N, Ordentlich A, Barak D, Bino T, Velan B, Shafferman A Ref: Biochemical Journal, 335:95, 1998 : PubMed
The role of the functional architecture of the human acetylcholinesterase (HuAChE) active centre in accommodating the non-covalent inhibitors tacrine and huperzine A, or the carbamates pyridostigmine and physostigmine, was analysed using 16 mutants of residues lining the active-centre gorge. Despite the structural diversity of the ligands, certain common properties of the complexes could be observed: (a) replacement of aromatic residues Tyr133, Tyr337 and especially Trp86, resulted in pronounced changes in stability of all the complexes examined; (b) effects due to replacements of the five other aromatic residues along the active-centre gorge, such as the acyl pocket (Phe295, Phe297) or at the peripheral anionic site (Tyr124, Trp286, Tyr341) were relatively small; (c) effects due to substitution of the carboxylic residues in the gorge (Glu202, Glu450) were moderate. These results and molecular modelling indicate that the aromatic side chains of residues Trp86, Tyr133 and Tyr337 form together a continuous 'aromatic patch' lining the wall of the active-centre gorge, allowing for the accommodation of the different ligands via multiple modes of interaction. Studies with HuAChE mutants carrying replacements at positions 86, 133 and 337 indicate that the orientations of huperzine A and tacrine in the HuAChE complexes in solution are significantly different from those observed in X-ray structures of the corresponding complexes with Torpedo californica AChE (TcAChE). These discrepancies may be explained in terms of structural differences between the complexes of HuAChE and TcAChE or, more likely, by the enhanced flexibility of the AChE active-centre gorge in solution as compared with the crystalline state.
Title: Contribution of aromatic moieties of tyrosine 133 and of the anionic subsite tryptophan 86 to catalytic efficiency and allosteric modulation of acetylcholinesterase Ordentlich A, Barak D, Kronman C, Ariel N, Segall Y, Velan B, Shafferman A Ref: Journal of Biological Chemistry, 270:2082, 1995 : PubMed
Substitution of Trp-86, in the active center of human acetylcholinesterase (HuAChE), by aliphatic but not by aromatic residues resulted in a several thousandfold decrease in reactivity toward charged substrate and inhibitors but only a severalfold decrease for noncharged substrate and inhibitors. The W86A and W86E HuAChE enzymes exhibit at least a 100-fold increase in the Michaelis-Menten constant or 100-10,000-fold increase in inhibition constants toward various charged inhibitors, as compared to W86F HuAChE or the wild type enzyme. On the other hand, replacement of Glu-202, the only acidic residue proximal to the catalytic site, by glutamine resulted in a nonselective decrease in reactivity toward charged and noncharged substrates or inhibitors. Thus, the quaternary nitrogen groups of substrates and other active center ligands, are stabilized by cation-aromatic interaction with Trp-86 rather than by ionic interactions, while noncharged ligands appear to bind to distinct site(s) in HuAChE. Analysis of the Y133F and Y133A HuAChE mutated enzymes suggests that the highly conserved Tyr-133 plays a dual role in the active center: (a) its hydroxyl appears to maintain the functional orientation of Glu-202 by hydrogen bonding and (b) its aromatic moiety maintains the functional orientation of the anionic subsite Trp-86. In the absence of aromatic interactions between Tyr-133 and Trp-86, the tryptophan acquires a conformation that obstructs the active site leading, in the Y133A enzyme, to several hundredfold decrease in rates of catalysis, phosphorylation, or in affinity to reversible active site inhibitors. It is proposed that allosteric modulation of acetylcholinesterase activity, induced by binding to the peripheral anionic sites, proceeds through such conformational change of Trp-86 from a functional anionic subsite state to one that restricts access of substrates to the active center.
Title: Role of tyrosine 337 in the binding of huperzine A to the active site of human acetylcholinesterase Ashani Y, Grunwald J, Kronman C, Velan B, Shafferman A Ref: Molecular Pharmacology, 45:555, 1994 : PubMed
Huperzine A (HUP), a natural, potent, 'slow,' reversible inhibitor of antiacetylcholinesterase (AChE), has been suggested to be superior to antiacetylcholinesterase drugs now being used for management of Alzheimer's disease. To delineate the binding site of human AChE (HuAChE) for HUP, the biochemical constants kon, koff, and Ki were determined for complexes formed between HUP and single-site (Y337F, Y337A, F295A, W286A, and E202Q) or double-site (F295L/F297V) mutants of recombinant HuAChE (rHuAChE). The kinetic and dissociation constants were compared with those obtained for wild-type rHuAChE and AChE from Torpedo californica. Results demonstrate that the inhibition of AChE by HUP occurs through association with residues located inside the active site 'gorge,' rather than at the rim of the gorge. Tyrosine at position 337 (Y337) is essential for inhibition of rHuAChE by HUP (Ki = 26 nM). An aromatic array constituted from residues Y337, F295, and probably W86 is likely to offer a multicontact subsite that interacts with the ammonium group and with both the exo-and endocyclic double bond moieties of HUP. Lack of the aromatic side chain in the position homologous to Y337 explains the poor inhibitory potency of HUP toward human butyrylcholinesterase (Ki > 20,000 nM). Replacement of the carboxylate-containing E202 by glutamine had only marginal effect on the stability of the complex formed between HUP and rHuAChE. The pH-rate profiles suggest that destabilization of the complex after proton gain cannot be attributed solely to protonation of E202. These findings are expected to establish HUP as a lead compound for the design of new anti-AChE drugs.
Several of the residues constituting the peripheral anionic site (PAS) in human acetylcholinesterase (HuAChE) were identified by a combination of kinetic studies with 19 single and multiple HuAChE mutants, fluorescence binding studies with the Trp-286 mutant, and by molecular modeling. Mutants were analyzed with three structurally distinct positively charged PAS ligands, propidium, decamethonium, and di(p-allyl-N-dimethylaminophenyl)pentane-3-one (BW284C51), as well as with selective active center inhibitors, hexamethonium and edrophonium. Single mutations of residues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341 resulted in up to 10-fold increase in inhibition constants for PAS ligands, whereas for multiple mutants up to 400-fold increase was observed. The 6th PAS element residue Asp-74 is unique in its ability to affect conformation of both the active site and the PAS (Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992) EMBO J. 11, 3561-3568) as demonstrated by the several hundred-fold increase in Ki for D74N inhibition by the bisquaternary ligands decamethonium and BW284C51. Based on these studies, singular molecular models for the various HuAChE inhibitor complexes were defined. Yet, for the decamethonium complex two distinct conformations were generated, accommodating the quaternary ammonium group by interactions with either Trp-286 or with Tyr-341. We propose that the PAS consists of a number of binding sites, close to the entrance of the active site gorge, sharing residues Asp-74 and Trp-286 as a common core. Binding of ligands to these residues may be the key to the allosteric modulation of HuAChE catalytic activity. This functional degeneracy is a result of the ability of the Trp-286 indole moiety to interact either via stacking, aromatic-aromatic, or via pi-cation attractions and the involvement of the carboxylate of Asp-74 in charge-charge or H-bond interactions.
Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.
Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.
Title: Acetylcholinesterase Catalysis - Protein Engineering Studies Shafferman A, Velan B Ref: In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases, (Shafferman, A. and Velan, B., Eds) Plenum Press, New York:165, 1992 : PubMed
