Liu Junjun, Hamza Adel, Zhan Chang-Guo
Key Laboratory of Pesticide and Chemical Biology of Ministry of Education, College of Chemistry, Central China Normal University, Wuhan 430079, People's Republic of China.
J Am Chem Soc. 2009 Aug 26;131(33):11964-75. doi: 10.1021/ja903990p.
The fundamental reaction mechanism of cocaine esterase (CocE)-catalyzed hydrolysis of (-)-cocaine and the corresponding free energy profile have been studied by performing pseudobond first-principles quantum mechanical/molecular mechanical free energy (QM/MM-FE) calculations. On the basis of the QM/MM-FE results, the entire hydrolysis reaction consists of four reaction steps, including the nucleophilic attack on the carbonyl carbon of (-)-cocaine benzoyl ester by the hydroxyl group of Ser117, dissociation of (-)-cocaine benzoyl ester, nucleophilic attack on the carbonyl carbon of (-)-cocaine benzoyl ester by water, and finally dissociation between the (-)-cocaine benzoyl group and Ser117 of CocE. The third reaction step involving the nucleophilic attack of a water molecule was found to be rate-determining, which is remarkably different from (-)-cocaine hydrolysis catalyzed by wild-type butyrylcholinesterase (BChE; where the formation of the prereactive BChE-(-)-cocaine complex is rate-determining) or its mutants containing Tyr332Gly or Tyr332Ala mutation (where the first chemical reaction step is rate-determining). Besides, the role of Asp259 in the catalytic triad of CocE does not follow the general concept of the "charge-relay system" for all serine esterases. The free energy barrier calculated for the rate-determining step of CocE-catalyzed hydrolysis of (-)-cocaine is 17.9 kcal/mol, which is in good agreement with the experimentally derived activation free energy of 16.2 kcal/mol. In the present study, where many sodium ions are present, the effects of counterions are found to be significant in determining the free energy barrier. The finding of the significant effects of counterions on the free energy barrier may also be valuable in guiding future mechanistic studies on other charged enzymes.
通过进行赝键第一性原理量子力学/分子力学自由能（QM/MM-FE）计算，研究了可卡因酯酶（CocE）催化水解（-）-可卡因的基本反应机理以及相应的自由能分布。基于QM/MM-FE结果，整个水解反应包括四个反应步骤，即Ser117的羟基对（-）-可卡因苯甲酰酯的羰基碳进行亲核进攻、（-）-可卡因苯甲酰酯解离、水对（-）-可卡因苯甲酰酯的羰基碳进行亲核进攻，最后（-）-可卡因苯甲酰基与CocE的Ser117解离。发现涉及水分子亲核进攻的第三步反应是速率决定步骤，这与野生型丁酰胆碱酯酶（BChE）催化的（-）-可卡因水解（其中预反应性BChE-（-）-可卡因复合物的形成是速率决定步骤）或其含有Tyr332Gly或Tyr332Ala突变的突变体（其中第一步化学反应是速率决定步骤）显著不同。此外，CocE催化三联体中Asp259的作用并不遵循所有丝氨酸酯酶的“电荷中继系统”的一般概念。CocE催化水解（-）-可卡因的速率决定步骤计算得到的自由能垒为17.9 kcal/mol，与实验得出的16.2 kcal/mol的活化自由能高度吻合。在本研究中存在许多钠离子的情况下，发现抗衡离子在决定自由能垒方面具有显著影响。抗衡离子对自由能垒有显著影响这一发现对于指导未来对其他带电荷酶的机理研究也可能具有重要价值。
