oxime interaction reactivation of tabun-inhibited human AChE. Rate constant 20-fold decreased for K027 and HLo-7. Unable to facilitate HI-6-mediated reactivation of tabun-hAChE Artursson_2009_Toxicology_265_108
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p.D74N Asp74Asn (p.D105N Asp105Asn in primary sequence with 31 amino-acids signal peptide) Substrate inhibition;Signal transduction Affects conformation of active site and PAS; strengthened heat or pressure stability ;decrease in VX stereoselectivity; reactivation of tabun-inhibited human AChE. Rate constant 20-fold decreased for K027 and HLo-7. Unable to facilitate HI-6-mediated reactivation of tabun-hAChE
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.
The reactivity of human acetylcholinesterase (HuAChE) toward the chemical warfare agent VX [O-ethyl S-[2-(diisopropylamino)ethyl] methyl-phosphonothioate] and its stereoselectivity toward the P(S)-enantiomer were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere nc-VX [O-ethyl S-(3-isopropyl-4-methyl-pentyl) methylphosphonothioate]. Stereoselectivity of the wild-type HuAChE toward VX(S) is manifested by a 115-fold higher bimolecular rate constant (1.4 x 10(8) min(-1) M(-1)) as compared to that of VX(R). HuAChE was also 12,500-fold more reactive toward VX(S) than toward nc-VX(S), demonstrating the significance of the polar interactions of the ammonium substituent to their overall affinity toward VX. Indeed, substitution of the cation-binding subsite residue Trp86 by alanine resulted in a decrease of three orders of magnitude in HuAChE reactivity toward both VX enantiomers, with only a marginal effect on the reactivity toward the enantiomers of nc-VX. These results demonstrate that accommodation of the charged moieties of both VX enantiomers depends predominantly on interactions with the aromatic moiety of Trp86. Yet, these interactions seem to limit the stereoselectivity toward the P(S)-enantiomer, which for charged methylphosphonates is much lower than for the noncharged analogs, like sarin or soman. Marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 by asparagine (D74N) practically abolished stereoselectivity toward VX(S) (a 130-fold decrease), while substitution which retained the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers originates predominantly from a different orientation of the charged leaving group with respect to residue Asp74. Such different orientations of the charged leaving group in the HuAChE adducts of the VX enantiomers seem to be a consequence of intramolecular interactions with the bulky phosphorus alkoxy group.
The origins of human acetylcholinesterase (HuAChE) reactivity toward the lethal chemical warfare agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) and its stereoselectivity toward the P(S)-VX enantiomer (VX(S)) were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere O-ethyl S-(3-isopropyl-4-methylpentyl) methylphosphonothioate (nc-VX) as well as echothiophate and its noncharged analogue. Reactivity of wild-type HuAChE toward VX(S) was 115-fold higher than that toward VX(R), with bimolecular rate constants of 1.4 x 10(8) and 1.2 x 10(6) min(-1) M(-1). HuAChE was also 12500-fold more reactive toward VX(S) than toward nc-VX(S). Substitution of the cation binding subsite residue Trp86 with alanine resulted in a 3 order of magnitude decrease in HuAChE reactivity toward both VX enantiomers, while this replacement had an only marginal effect on the reactivity toward the enantiomers of nc-VX and the noncharged echothiophate. These results attest to the critical role played by Trp86 in accommodating the charged moieties of both VX enantiomers. A marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 with asparagine (D74N) practically abolished stereoselectivity toward VX(S) (130-fold decrease), while a substitution which retains the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers is mainly a result of a different interaction of the charged leaving group with Asp74.
Title: Pressure and heat inactivation of recombinant human acetylcholinesterase. Importance of residue E202 for enzyme stability Clery-Barraud C, Ordentlich A, Grosfeld H, Shafferman A, Masson P Ref: European Journal of Biochemistry, 269:4297, 2002 : PubMed
The effects of pressure on structure and activity of recombinant human acetylcholinesterase (rHuAChE) were investigated up to a pressure of 300 MPa using gel electrophoresis under elevated hydrostatic pressure, fluorescence of bound 8-anilinonaphthalene-1-sulfonate (ANS) and activity measurements following exposure to high pressure. Study of wild-type enzyme and three single mutants (D74N, E202Q, E450A) and one sextuple mutant (E84Q/E292A/D349N/E358Q/E389Q/D390N) showed that pressure exerts a differential action on wild-type rHuAChE and its mutants, allowing estimation of the contribution of carboxylic amino acid side-chains to enzyme stability. Mutation of negatively charged residues D74 and E202 by polar side-chains strengthened heat or pressure stability. The mutation E450A and the sextuple mutation caused destabilization of the enzyme to pressure. Thermal inactivation data on mutants showed that all of them were stabilized against temperature. In conclusion, pressure and thermal stability of mutants provided evidence that the residue E202 is a determinant of structural and functional stability of HuAChE.
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: Aging of phosphylated human acetylcholinesterase: catalytic processes mediated by aromatic and polar residues of the active centre Shafferman A, Ordentlich A, Barak D, Stein D, Ariel N, Velan B Ref: Biochemical Journal, 318:833, 1996 : PubMed
We have examined the effects of 11 substitutions of active centre gorge residues of human acetylcholinesterase (HuAChE) on the rates of phosphonylation by 1,2,2-trimethylpropyl methyl-phosphonofluoridate (soman) and the aging of the resulting conjugates. The rates of phosphonylation were reduced to as little as one-seventieth, mainly in mutants of the hydrogen-bond network (Glu-202, Glu-450, Tyr-133). These recombinant enzymes as well as the F338A, W86A, W86F and D74N mutant HuAChEs varied in their resistance to aging (15-3300-fold relative to the wild type). The most dramatic resistance to aging was observed for the phosphonyl conjugate of the mutant W86A enzyme (1850-3300-fold relative to the wild type). It is proposed that Trp-86 contributes to the aging process by stabilizing the evolving carbonium ion on the 1,2,2-trimethylpropyl moiety, via charge-pi interaction. The rate-enhancing effect of Trp-86 provides a rationale for the unique facility of aging in soman-inhibited cholinesterases, compared with the corresponding conjugates in other serine hydrolases. Replacements of Glu-202 by aspartic acid, glutamine or alanine residues resulted in a similar (1/130-1/300) decrease of the rates of aging. A comparable decrease was also observed for the conjugate of the F338A mutant. These results, and the similar pH dependence of aging rates for the wild-type and E202Q and F338A mutant HuAChEs, indicate that Glu-202 is not involved in proton transfer to the phosphonyl moiety. On the basis of these findings and of molecular modelling we suggest that Glu-202 and Phe-338 contribute to the aging process by stabilizing the imidazolium of the catalytic triad His-447 via charge-charge and charge-pi interactions respectively, thereby facilitating an oxonium formation on the phosphonyl moiety.
Conformational mobility of the surface omega loop (Cys-69-Cys-96) in human acetylcholinesterase (HuAChE) was recently implicated in substrate accessibility to the active center and in the mechanism of allosteric modulation of enzymatic activity. We therefore generated and kinetically evaluated the following modifications or replacements in HuAChE: (a) residues at the loop ends, (b) residues involved in putative hydrogen-bond interactions within the loop and between the loop and the protein core, (c) ChEs conserved proline residues within the loop and (d) a deletion of a conserved segment of 5 residues. All the residue replacements, including those of the prolines, had either limited or no effect on enzyme reactivity. These results suggest that unlike the case of lipase, the omega loop in the HuAChE is not involved in large lid-like displacements. In cases where modifications of the loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp-86 supporting the proposed coupling between the structure of the omega loop and the positioning of the Trp-86 indole moiety, in catalytic activity and in allosterism.
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.
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
