Free Energy Perturbation (FEP) Simulation on the Transition States of Cocaine Hydrolysis Catalyzed by Human Butyrylcholinesterase and Its Mutants

摘要

A novel computational protocol based on free energy perturbation (FEP) simulations on both the free enzyme and transition state structures has been developed and tested to predict the mutation-caused shift of the free energy change from the free enzyme to the rate-determining transition state for human butyrylcholinesterase (BChE)-catalyzed hydrolysis of (-)-cocaine. The calculated shift, denoted by ΔΔG(1 → 2), of such kind of free energy change determines the catalytic efficiency (k_(cat)/K_M) change caused by the simulated mutation transforming enzyme 1 to enzyme 2. By using the FEP-based computational protocol, the ΔΔG(1 → 2) values for the mutations A328W/Y332A → A328W/Y332G and A328W/Y332G → A328W/Y332G/A199S were calculated to be -0.22 and -1.94 kcal/mol, respectively. The calculated ΔΔG(1 → 2) values predict that the change from the A328W/Y332A mutant to the A328W/Y332G mutant should slightly improve the catalytic efficiency and that the change from the A328W/Y332G mutant to the A328W/Y332G/A199S mutant should significantly improve the catalytic efficiency of the enzyme for the (-)-cocaine hydrolysis. The predicted catalytic efficiency increases are supported by the experimental data showing that k_(cat)/K_M = 8.5 × 10~6, 1.4 × 10~7, and 7.2 × 10~7 min~(-1) M~(-1) for the A328W/Y332A, A328W/ Y332G, and A328W/Y332G/A199S mutants, respectively. The qualitative agreement between the computational and experimental data suggests that the FEP simulations may provide a promising protocol for rational design of high-activity mutants of an enzyme. The general computational strategy of the FEP simulation on a transition state can be used to study the effects of a mutation on the activation free energy for any enzymatic reaction.