The A199S/F227A/S287G/A328W/Y332G mutant of human butyrylcholineserase(BChE)catalyzes the hydrolysis of cocaine with high efficiency.It is not clear whether or not this hydrolyase will enhance the hydrolysis of neurotransmitter acetylcholine(ACh),which is an important neurotransmitter in the impulse transmission.Herein we studied the reaction pathway of ACh hydrolysis catalyzed by this mutant and the corresponding free energy profile by performing pseudobond first-principles quantum mechanical/molecular mechanical-free energy(QM/MM-FE)calculations.Based on the QM/MM-FE results,the catalytic process consists of four steps,including the nucleophilic attack on the carbonyl carbon of ACh,the dissociation of choline ester,nucleophilic attack of a water molecule on the carboxyl carbon of substrate and dissociation between the carboxyl carbon of substrate and hydroxyl oxygen of Ser198 side chain.The second reaction step is rate-determining,which is slightly different from the previous known mechanism for ACh hydrolysis catalyzed by wild-type BChE,where the first reaction step was found to be rate-determining.The present theoretical and experimental studies suggest that catalytic constant for the ACh hydrolysis catalyzed by the A199S/F227A/S287G/A328W/Y332G BChE is smaller than that for the ACh catalyzed by the wild-type BChE.The calculated free energy barrier associated with rate-determining step is~14.3 kcal/mol,which is in good agreement with the experimentally derived activation free energy of~13.6 kcal/mol.Combined with the results from previous studies on the hydrolysis of(-)-cocaine catalyzed by this hydrolyase,the present results demonstrate that the oxyanion hole of this hydrolyase play an important role of in improving its selectivity against(-)-cocaine.