Journal of the American Chemical Society, Vol.129, No.44, 13537-13543, 2007
Free energy perturbation (FEP) simulation on the transition states of cocaine hydrolysis catalyzed by human butyrylcholinesterase and its mutants
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 mutationcaused 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 Delta Delta G(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 Delta Delta G(l - 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 Delta Delta G(l - 2) values predict that the change from the A328WN332A 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 x 10(6), 1.4 x 10(7), and 7.2 x 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.