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Stereoselectivity toward VX Is Determined by Interactions with Residues of the Acyl Pocket as Well as of the Peripheral Anionic Site of AChE

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journal contribution
posted on 07.09.2004, 00:00 by Arie Ordentlich, Dov Barak, Gali Sod-Moriah, Dana Kaplan, Dana Mizrahi, Yoffi Segall, Chanoch Kronman, Yishai Karton, Arie Lazar, Dino Marcus, Baruch Velan, Avigdor Shafferman
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 PS-VX enantiomer (VXS) 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 VXS was 115-fold higher than that toward VXR, with bimolecular rate constants of 1.4 × 108 and 1.2 × 106 min-1 M-1. HuAChE was also 12500-fold more reactive toward VXS than toward nc-VXS. 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 VXS 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 VXS (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.