title: 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 DeltaDeltaG(1 --> 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 --> 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 DeltaDeltaG(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 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.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 725 Rose Street, Lexington, KY 40536, USA.
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Pan
authors.forename: Daquan
authors.initials: D
authors.lastname: Gao
authors.forename: Wenchao
authors.initials: W
authors.lastname: Yang
authors.forename: Hoon
authors.initials: H
authors.lastname: Cho
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites; D002091:Butyrylcholinesterase / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D003042:Cocaine / Q000737:chemistry*; D003198:Computer Simulation; D006801:Humans; D006868:Hydrolysis; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D013237:Stereoisomerism; D013816:Thermodynamics
chemical_list: D002091:Butyrylcholinesterase; D003042:Cocaine