Reaction Pathway and Free Energy Profile for Cocaine Hydrolase-Catalyzed Hydrolysis of (-)-Cocaine.
Reaction Pathway and Free Energy Profile for Cocaine Hydrolase-Catalyzed Hydrolysis of (-)-Cocaine.
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DOI：
10.1021/ct200810d
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发表时间：
2012-04-10
影响因子：
5.5
通讯作者：
Zhan, Chang-Guo
中科院分区：
文献类型：
--
作者：
Liu, Junjun;Zhan, Chang-Guo
采用第一性原理赝键量子力学/分子力学（QM/MM）自由能（FE）方法，研究了新近发现的最有效的可卡因水解酶--人丁酰胆碱酯酶（BChE）突变体A199 S/F227 A/S287 G/A328 W/Y332 G催化（-）-可卡因水解的反应途径及其自由能分布.根据QM/MM-FE结果，催化水解过程包括四个主要反应步骤，包括S198的羟基对（-）-可卡因苯甲酰基酯的羰基碳的亲核攻击，（-）-可卡因苯甲酰基酯的解离，水对（-）-可卡因苯甲酰基酯的羰基碳的亲核攻击，最后是（-）-可卡因苯甲酰基和酶的S198之间的解离。第二步反应是速率决定的。计算的自由能势垒与过渡态的速率决定步骤是~15.0千卡/摩尔，这是在良好的协议与实验导出的活化自由能~14.7千卡/摩尔。从本研究中获得的机制的见解将是有价值的合理设计更积极的可卡因水解酶对（-）-可卡因。特别是，未来旨在进一步提高该酶对（-）-可卡因的催化活性的努力应集中于稳定第二反应步骤的过渡态，在该步骤中（-）-可卡因的苯甲酰酯解离。
Reaction pathway of (−)-cocaine hydrolysis catalyzed by our recently discovered most efficient cocaine hydrolase, which is the A199S/F227A/S287G/A328W/Y332G mutant of human butyrylcholinesterase (BChE), and the corresponding free energy profile have been studied by performing first-principles pseudobond quantum mechanical/molecular mechanical (QM/MM)-free energy (FE) calculations. Based on the QM/MM-FE results, the catalytic hydrolysis process consists of four major reaction steps, including the nucleophilic attack on carbonyl carbon of (−)-cocaine benzoyl ester by hydroxyl group of S198, dissociation of (−)-cocaine benzoyl ester, nucleophilic attack on carbonyl carbon of (−)-cocaine benzoyl ester by water, and finally the dissociation between (−)-cocaine benzoyl group and S198 of the enzyme. The second reaction step is rate-determining. The calculated free energy barrier associated with the transition state for the rate-determining step is ~15.0 kcal/mol, which is in excellent agreement with the experimentally-derived activation free energy of ~14.7 kcal/mol. The mechanistic insights obtained from the present study will be valuable for rational design of more active cocaine hydrolase against (−)-cocaine. In particular, future efforts aiming at further increasing the catalytic activity of the enzyme against (−)-cocaine should focus on stabilization of the transition state for the second reaction step in which the benzoyl ester of (−)-cocaine dissociates.
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DOI：
10.1073/pnas.95.17.10176
发表时间：
1998-08-18
影响因子：
11.1
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