Ordentlich Arie, Barak Dov, Sod-Moriah Gali, Kaplan Dana, Mizrahi Dana, Segall Yoffi, Kronman Chanoch, Karton Yishai, Lazar Arie, Marcus Dino, Velan Baruch, Shafferman Avigdor
Department of Biochemistry & Molecular Genetics, Israel Institute for Biological Research, Ness-Ziona 74100, Israel.
Chem Biol Interact. 2005 Dec 15;157-158:191-8. doi: 10.1016/j.cbi.2005.10.026. Epub 2005 Nov 10.
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.
通过研究人乙酰胆碱酯酶（HuAChE）及其突变衍生物对VX[O-乙基-S-[2-(二异丙基氨基)乙基]甲基硫代磷酸酯]的纯化对映体及其不带电荷的等排体nc-VX[O-乙基-S-(3-异丙基-4-甲基戊基)甲基硫代磷酸酯]的反应活性，研究了HuAChE对化学战剂VX的反应活性及其对P(S)-对映体的立体选择性。野生型HuAChE对VX(S)的立体选择性表现为其双分子速率常数（1.4×10⁸ min⁻¹ M⁻¹）比VX(R)高115倍。HuAChE对VX(S)的反应活性也比对nc-VX(S)高12500倍，这表明铵取代基的极性相互作用对其与VX的整体亲和力具有重要意义。事实上，将阳离子结合亚位点残基Trp86替换为丙氨酸会导致HuAChE对两种VX对映体的反应活性降低三个数量级，而对nc-VX对映体的反应活性影响很小。这些结果表明，两种VX对映体带电部分的容纳主要取决于与Trp86芳香部分的相互作用。然而，这些相互作用似乎限制了对P(S)-对映体的立体选择性，对于带电的甲基膦酸酯来说，其立体选择性比对不带电的类似物（如沙林或梭曼）要低得多。在酰基口袋处替换Phe295（F295A和F295A/F297A）后，观察到对VX(S)的立体选择性显著降低。将外周阴离子位点（PAS）残基Asp74替换为天冬酰胺（D74N）实际上消除了对VX(S)的立体选择性（降低了130倍），而在74位保留负电荷的替换（D74E）则没有影响。动力学研究和分子模拟结果表明，对VX对映体的不同反应活性主要源于带电离去基团相对于残基Asp74的不同取向。在VX对映体的HuAChE加合物中，带电离去基团的这种不同取向似乎是与庞大的磷烷氧基分子内相互作用的结果。
