Ordentlich A, Barak D, Kronman C, Ariel N, Segall Y, Velan B, Shafferman A
Department of Biochemistry, Israel Institute for Biological Research, Ness-Ziona.
J Biol Chem. 1996 May 17;271(20):11953-62. doi: 10.1074/jbc.271.20.11953.
The role of the functional architecture of human acetylcholinesterase (HuAChE) active center in facilitating reactions with organophosphorus inhibitors was examined by a combination of site-directed mutagenesis and kinetic studies of phosphorylation with organophosphates differing in size of their alkoxy substituents and in the nature of the leaving group. Replacements of residues Phe-295 and Phe-297, constituting the HuAChE acyl pocket, increase up to 80-fold the reactivity of the enzymes toward diisopropyl phosphorofluoridate, diethyl phosphorofluoridate, and p-nitrophenyl diethyl phosphate (paraoxon), indicating the role of this subsite in accommodating the phosphate alkoxy substituent. On the other hand, a decrease of up to 160-fold in reactivity was observed for enzymes carrying replacements of residues Tyr-133, Glu-202, and Glu-450, which are constituents of the hydrogen bond network in the HuAChE active center, which maintains its unique functional architecture. Replacement of residues Trp-86, Tyr-337, and Phe-338 in the alkoxy pocket affected reactivity toward diisopropyl phosphorofluoridate and paraoxon, but to a lesser extent that toward diethyl phosphorofluoridate, indicating that both the alkoxy substituent and the p-nitrophenoxy leaving group interact with this subsite. In all cases the effects on reactivity toward organophosphates, demonstrated in up to 10,000-fold differences in the values of bimolecular rate constants, were mainly a result of altered affinity of the HuAChE mutants, while the apparent first order rate constants of phosphorylation varied within a narrow range. This finding indicates that the main role of the functional architecture of HuAChE active center in phosphorylation is to facilitate the formation of enzyme-inhibitor Michaelis complexes and that this affinity, rather than the nucleophilic activity of the enzyme catalytic machinery, is a major determinant of HuAChE reactivity toward organophosphates.
通过定点诱变以及对具有不同烷氧基取代基大小和离去基团性质的有机磷酸酯进行磷酸化动力学研究相结合的方法，研究了人乙酰胆碱酯酶（HuAChE）活性中心的功能结构在促进与有机磷抑制剂反应中的作用。构成HuAChE酰基口袋的苯丙氨酸-295和苯丙氨酸-297残基被取代后，酶对氟磷酸二异丙酯、氟磷酸二乙酯和对硝基苯基二乙基磷酸酯（对氧磷）的反应活性提高了80倍，这表明该亚位点在容纳磷酸烷氧基取代基方面的作用。另一方面，对于携带酪氨酸-133、谷氨酸-202和谷氨酸-450残基被取代的酶，观察到反应活性降低了160倍，这些残基是HuAChE活性中心氢键网络的组成部分，该网络维持其独特的功能结构。烷氧基口袋中的色氨酸-86、酪氨酸-337和苯丙氨酸-338残基被取代影响了对氟磷酸二异丙酯和对氧磷的反应活性，但对对氟磷酸二乙酯的反应活性影响较小，这表明烷氧基取代基和对硝基苯氧基离去基团都与该亚位点相互作用。在所有情况下，对有机磷酸酯反应活性的影响，在双分子速率常数的值上表现出高达10000倍的差异，主要是HuAChE突变体亲和力改变的结果，而磷酸化的表观一级速率常数在狭窄范围内变化。这一发现表明，HuAChE活性中心功能结构在磷酸化中的主要作用是促进酶-抑制剂米氏复合物的形成，并且这种亲和力，而不是酶催化机制的亲核活性，是HuAChE对有机磷酸酯反应活性的主要决定因素。
