title: Stereoselectivity toward VX is determined by interactions with residues of the acyl pocket as well as of the peripheral anionic site of AChE.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona, 74100 Israel.
authors.forename: Arie
authors.initials: A
authors.lastname: Ordentlich
authors.forename: Dov
authors.initials: D
authors.lastname: Barak
authors.forename: Gali
authors.initials: G
authors.lastname: Sod-Moriah
authors.forename: Dana
authors.initials: D
authors.lastname: Kaplan
authors.forename: Dana
authors.initials: D
authors.lastname: Mizrahi
authors.forename: Yoffi
authors.initials: Y
authors.lastname: Segall
authors.forename: Chanoch
authors.initials: C
authors.lastname: Kronman
authors.forename: Yishai
authors.initials: Y
authors.lastname: Karton
authors.forename: Arie
authors.initials: A
authors.lastname: Lazar
authors.forename: Dino
authors.initials: D
authors.lastname: Marcus
authors.forename: Baruch
authors.initials: B
authors.lastname: Velan
authors.forename: Avigdor
authors.initials: A
authors.lastname: Shafferman
mesh_terms: D000110:Acetylcholinesterase / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000838:Anions / Q000737:chemistry; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002794:Choline / Q000235:genetics; D002800:Cholinesterase Inhibitors / Q000737:chemistry*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D009946:Organothiophosphorus Compounds / Q000737:chemistry*; D011485:Protein Binding / Q000235:genetics; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics
chemical_list: D000110:Acetylcholinesterase; D000838:Anions; D001224:Aspartic Acid; D002794:Choline; D002800:Cholinesterase Inhibitors; D007527:Isoenzymes; D009946:Organothiophosphorus Compounds; C009680:VX-agent