title: Aging of phosphylated human acetylcholinesterase: catalytic processes mediated by aromatic and polar residues of the active centre.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Israel Institute for Biological Research, Ness-Ziona, Israel.
authors.forename: A
authors.initials: A
authors.lastname: Shafferman
authors.forename: A
authors.initials: A
authors.lastname: Ordentlich
authors.forename: D
authors.initials: D
authors.lastname: Barak
authors.forename: D
authors.initials: D
authors.lastname: Stein
authors.forename: N
authors.initials: N
authors.lastname: Ariel
authors.forename: B
authors.initials: B
authors.lastname: Velan
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000375:Aging / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D004795:Enzyme Stability; D006801:Humans; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017976:Phosphorus Acids / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D017976:Phosphorus Acids; D011994:Recombinant Proteins; D000110:Acetylcholinesterase