Reaction Pathway and Free Energy Profile for Cocaine Hydrolase-Catalyzed Hydrolysis of (-)-Cocaine.

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.