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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 DeltaDeltaG(1 --&gt; 2), of such kind of free energy change determines the catalytic efficiency (kcat/KM) change caused by the simulated mutation transforming enzyme 1 to enzyme 2. By using the FEP-based computational protocol, the DeltaDeltaG(1 --&gt; 2) values for the mutations A328W/Y332A --&gt; A328W/Y332G and A328W/Y332G --&gt; A328W/Y332G/A199S were calculated to be -0.22 and -1.94 kcal/mol, respectively. The calculated DeltaDeltaG(1 --&gt; 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 kcat/KM = 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.

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