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

Abstract

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 highactivity 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.

Design, Preparation, and Characterization of High-Activity Mutants of Human Butyrylcholinesterase Specific for Detoxification of Cocaine

“…Therefore, starting from the A328W/Y332A or A328W/ Y332G mutant, rational design of BChE mutants against (Ϫ)-cocaine has been focused on decreasing the energy barrier for the first reaction step without significantly affecting the other reaction steps. We have developed unique computational strategies and protocols based on the virtual screening of rate-determining transition states of the enzymatic reaction to design enzyme mutants with improved catalytic activity (Pan et al, 2005(Pan et al, , 2007(Pan et al, , 2008Zheng et al, 2008Zheng et al, , 2010Yang et al, 2009). The computational design was followed by in vitro experiments, including site-directed mutagenesis, protein expression, and fast enzyme activity screening using the culture medium.…”

“…The computational design was followed by in vitro experiments, including site-directed mutagenesis, protein expression, and fast enzyme activity screening using the culture medium. The integrated computational-experimental studies have led to the discovery of some BChE mutants with a significantly improved catalytic efficiency against (Ϫ)-cocaine (Pan et al, 2005(Pan et al, , 2007(Pan et al, , 2008Zheng et al, 2008;Yang et al, 2009;Yang et al, 2010). One of our designed and discovered high-activity mutants of human BChE (i.e., the A199S/S287G/A328W/Y332G mutant) (Pan et al, 2005) has been validated by an independent group of scientists who concluded that this mutant is "a true cocaine hydrolase with a catalytic efficiency that is 1000-fold greater than wild-type BChE.…”

Thermostable Variants of Cocaine Esterase for Long-Time Protection against Cocaine Toxicity

“…The classic central nervous system receptor-antagonist approach has failed to yield an anticocaine therapeutic, but we developed a proof of principle for a peripheral blocker to accelerate cocaine metabolism in the circulation (Landry et al, 1993;Mets et al, 1998), producing biologically inactive metabolites via hydrolysis of cocaine benzoyl ester (Gorelick, 1997;Zhan et al, 2003;Meijler et al, 2005;Pan et al, 2005Pan et al, , 2007Rogers et al, 2005;Zheng et al, 2008). The bacterial cocaine esterase (CocE) (Bresler et al, 2000), the most efficient native cocaine hydrolase yet identified , is particularly promising.…”

Computational Studies of Glycoside, Carboxylic Ester, and Thioester Hydrolase Mechanisms: A Review

“…Pan et al confirmed some of these predictions by employing combined MD and free-energy perturbation on human BChE in the free state and at the TS with (−)-cocaine substrate. 150 They determined ΔΔG(1 → 2) values between the two states, allowing them to calculate k cat /K M between simulated A328W/Y332A and A328W/Y332G mutants and between simulated A328W/Y332G and A328W/Y332G/ A199S mutants. They found that their predicted kinetic efficiencies agreed with experimental values, the first pair of mutations yielding a small increase and the second pair a bigger increase.…”