title: Free-energy perturbation simulation on transition states and redesign of butyrylcholinesterase.
abstract: It is recognized that an ideal anti-cocaine treatment is to accelerate cocaine metabolism by producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., butyrylcholinesterase (BChE)-catalyzed hydrolysis of cocaine. BChE mutants with a higher catalytic activity against (-)-cocaine are highly desired for use as an exogenous enzyme in humans. To develop a rational design for high-activity mutants, we carried out free-energy perturbation (FEP) simulations on various mutations of the transition-state structures in addition to the corresponding free-enzyme structures by using an extended FEP procedure. The FEP simulations on the mutations of both the free-enzyme and transition-state structures allowed us to calculate the mutation-caused shift of the free-energy change from the free enzyme (BChE) to the transition state, and thus to theoretically predict the mutation-caused shift of the catalytic efficiency (k(cat)/K(M)). The computational predictions are supported by the kinetic data obtained from the wet experiments, demonstrating that the FEP-based computational design approach is promising for rational design of high-activity mutants of an enzyme. One of the BChE mutants designed and discovered in this study has an approximately 1800-fold improved catalytic efficiency against (-)-cocaine compared to wild-type BChE. The high-activity mutant may be therapeutically valuable.
authors.affiliation: Key Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry, Central China Normal University, Wuhan, PR China.
authors.forename: Wenchao
authors.initials: W
authors.lastname: Yang
authors.forename: Yongmei
authors.initials: Y
authors.lastname: Pan
authors.forename: Fang
authors.initials: F
authors.lastname: Zheng
authors.forename: Hoon
authors.initials: H
authors.lastname: Cho
authors.forename: Hsin-Hsiung
authors.initials: HH
authors.lastname: Tai
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
mesh_terms: D000465:Algorithms; D002091:Butyrylcholinesterase / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D003042:Cocaine / Q000737:chemistry; D003198:Computer Simulation; D017076:Computer-Aided Design; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry; D009154:Mutation; D015202:Protein Engineering
chemical_list: D050505:Mutant Proteins; D002091:Butyrylcholinesterase; D003042:Cocaine