Liu_2012_J.Chem.Theory.Comput_8_1426

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.