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. 1996 Sep 15;318(Pt 3):833–840. doi: 10.1042/bj3180833

Aging of phosphylated human acetylcholinesterase: catalytic processes mediated by aromatic and polar residues of the active centre.

A Shafferman 1, A Ordentlich 1, D Barak 1, D Stein 1, N Ariel 1, B Velan 1
PMCID: PMC1217693  PMID: 8836126

Abstract

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.

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Selected References

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