title: Reaction mechanism for cocaine esterase-catalyzed hydrolyses of (+)- and (-)-cocaine: unexpected common rate-determining step.
abstract: First-principles quantum mechanical/molecular mechanical free energy calculations have been performed to examine the catalytic mechanism for cocaine esterase (CocE)-catalyzed hydrolysis of (+)-cocaine in comparison with CocE-catalyzed hydrolysis of (-)-cocaine. It has been shown that the acylation of (+)-cocaine consists of nucleophilic attack of the hydroxyl group of Ser117 on the carbonyl carbon of (+)-cocaine benzoyl ester and the dissociation of (+)-cocaine benzoyl ester. The first reaction step of deacylation of (+)-cocaine, which is identical to that of (-)-cocaine, is rate-determining, indicating that CocE-catalyzed hydrolyses of (+)- and (-)-cocaine have a common rate-determining step. The computational results predict that the catalytic rate constant of CocE against (+)-cocaine should be the same as that of CocE against (-)-cocaine, in contrast with the remarkable difference between human butyrylcholinesterase-catalyzed hydrolyses of (+)- and (-)-cocaine. The prediction has been confirmed by experimental kinetic analysis on CocE-catalyzed hydrolysis of (+)-cocaine in comparison with CocE-catalyzed hydrolysis of (-)-cocaine. The determined common rate-determining step indicates that rational design of a high-activity mutant of CocE should be focused on the first reaction step of the deacylation. Furthermore, the obtained mechanistic insights into the detailed differences in the acylation between the (+)- and (-)-cocaine hydrolyses provide indirect clues for rational design of amino acid mutations that could more favorably stabilize the rate-determining transition state in the deacylation and, thus, improve the catalytic activity of CocE. This study provides a valuable mechanistic base for rational design of an improved esterase for therapeutic treatment of cocaine abuse.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, Kentucky 40536, USA.
authors.forename: Junjun
authors.initials: J
authors.lastname: Liu
authors.forename: Xinyun
authors.initials: X
authors.lastname: Zhao
authors.forename: Wenchao
authors.initials: W
authors.lastname: Yang
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
mesh_terms: D055162:Biocatalysis; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D003042:Cocaine / Q000737:chemistry*; D006868:Hydrolysis; D008958:Models, Molecular; D015394:Molecular Structure; D011789:Quantum Theory; D013237:Stereoisomerism
chemical_list: D002265:Carboxylic Ester Hydrolases; C019921:cocaine esterase; D003042:Cocaine