Reaction pathway and free energy profile for cocaine hydrolase-catalyzed hydrolysis of (-)-cocaine

The 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. On the basis of the QM/MM-FE results, the catalytic hydrolysis process consists of four major reaction steps, including the nucleophilic attack on the carbonyl carbon of the (-)-cocaine benzoyl ester by the hydroxyl group of S198, dissociation of the (-)-cocaine benzoyl ester, nucleophilic attack on the carbonyl carbon of the (-)-cocaine benzoyl ester by water, and finally the dissociation between the (-)-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 the 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.