One- and Two-Proton Transfer Mechanisms Coexist in One Active Site
One- and Two-Proton Transfer Mechanisms Coexist in One Active Site
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一质子和二质子转移机制共存于一个活性位点
DOI：
10.1021/acs.jpcb.0c04445
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发表时间：
2020
影响因子：
3.3
通讯作者：
Wang Xia
中科院分区：
文献类型：
--
作者：
Zhao Yueqi;Dong Huaikun;Ren Jing;Song Jiali;Yao Jianzhuang;Gao Juan;Jiang Cheng-Shi;Wang Xia
酸性苯并烯-S-甲基是目前商业化程度最高的系统获得性抗性植物激活剂之一。然而，水杨酸结合蛋白2(SABP2)催化其活化(水解)的机制仍不清楚。通过大量的计算和实验研究，包括QM/MM模拟、电荷转移分析、小分子合成和生化分析，研究了SABP2催化ASM水解的基本机理。在这里，我们报告了混杂的SABP2对不同的底物表现出不同的催化机理。SABP2采用两质子转移机制，关键中间体通过电荷转移稳定；SABP2催化ASM类似物1，2，3-苯并噻二唑-7-羧酸乙酯(BTM)的水解，采用单质子转移机制，经典的四面体中间体通过静电作用稳定。SABP2对ASM和BTM的酯酶活性的高效液相分析结果与我们的计算结果相吻合，表明所获得的计算机理见解是合理的。所获得的机理不仅是对酶催化混杂理论的重要补充，也为下一代植物SAR激活剂的设计提供了可能的策略。
Acibenzolar-S-methyl (ASM) is one of the most successfully commercialized plant activators of the systemic acquired resistance (SAR). However, its activation (hydrolysis) mechanism catalyzed by the salicylic acid binding protein 2 (SABP2) remains elusive. The fundamental catalytic mechanism of the SABP2-catalyzed hydrolysis of the ASM had been investigated by extensive computational and experimental studies, including QM/MM simulations, charge transfer analysis, small-molecule synthesis, and biochemical assays. Here we report that the promiscuous SABP2 shows different catalytic mechanisms toward different substrates. To catalyze the ASM hydrolysis, the SABP2 uses a two-proton transfer mechanism, and the key intermediate is stabilized by the charge transfer effect; to catalyze the ethyl 1,2,3-benzothiadiazole-7-carboxylate (BTM, an ASM analogue) hydrolysis, the SABP2 applies the one-proton transfer mechanism, and the classic tetrahedral intermediate is stabilized by the electrostatic effect. The HPLC analyses of the SABP2 esterase activities toward the ASM and the BTM show comparable results with our computaional results, suggesting that the obtained computational mechanism insights are reasonable. The obtained mechanism is not only an important supplement to the theory of enzymes’ catalytic promiscuity, but it also contributes a possible strategy for the design of next generation plant SAR activators.