title: Kinetic characterization of a cocaine hydrolase engineered from mouse butyrylcholinesterase.
abstract: Mouse butyrylcholinesterase (mBChE) and an mBChE-based cocaine hydrolase (mCocH, i.e. the A¹⁹⁹S/S²²⁷A/S²⁸⁷G/A³²⁸W/Y³³²G mutant) have been characterized for their catalytic activities against cocaine, i.e. naturally occurring (-)-cocaine, in comparison with the corresponding human BChE (hBChE) and an hBChE-based cocaine hydrolase (hCocH, i.e. the A¹⁹⁹S/F²²⁷A/S²⁸⁷G/A³²⁸W/Y³³²G mutant). It has been demonstrated that mCocH and hCocH have improved the catalytic efficiency of mBChE and hBChE against (-)-cocaine by ~8- and ~2000-fold respectively, although the catalytic efficiencies of mCocH and hCocH against other substrates, including acetylcholine (ACh) and butyrylthiocholine (BTC), are close to those of the corresponding wild-type enzymes mBChE and hBChE. According to the kinetic data, the catalytic efficiency (k(cat)/K(M)) of mBChE against (-)-cocaine is comparable with that of hBChE, but the catalytic efficiency of mCocH against (-)-cocaine is remarkably lower than that of hCocH by ~250-fold. The remarkable difference in the catalytic activity between mCocH and hCocH is consistent with the difference between the enzyme-(-)-cocaine binding modes obtained from molecular modelling. Further, both mBChE and hBChE demonstrated substrate activation for all of the examined substrates [(-)-cocaine, ACh and BTC] at high concentrations, whereas both mCocH and hCocH showed substrate inhibition for all three substrates at high concentrations. The amino-acid mutations have remarkably converted substrate activation of the enzymes into substrate inhibition, implying that the rate-determining step of the reaction in mCocH and hCocH might be different from that in mBChE and hBChE.
authors.affiliation: *Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, U.S.A.
authors.forename: Xiabin
authors.initials: X
authors.lastname: Chen
authors.affiliation: *Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, U.S.A.
authors.forename: Xiaoqin
authors.initials: X
authors.lastname: Huang
authors.affiliation: †Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, U.S.A.
authors.forename: Liyi
authors.initials: L
authors.lastname: Geng
authors.affiliation: *Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, U.S.A.
authors.forename: Liu
authors.initials: L
authors.lastname: Xue
authors.affiliation: *Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, U.S.A.
authors.forename: Shurong
authors.initials: S
authors.lastname: Hou
authors.affiliation: *Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, U.S.A.
authors.forename: Xirong
authors.initials: X
authors.lastname: Zheng
authors.affiliation: †Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, U.S.A.
authors.forename: Stephen
authors.initials: S
authors.lastname: Brimijoin
authors.affiliation: *Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, U.S.A.
authors.forename: Fang
authors.initials: F
authors.lastname: Zheng
authors.affiliation: *Molecular Modeling and Biopharmaceutical Center and Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, U.S.A.
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
mesh_terms: D000109:Acetylcholine / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D055162:Biocatalysis; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002092:Butyrylthiocholine / Q000737:chemistry / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003042:Cocaine / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D002092:Butyrylthiocholine; D002265:Carboxylic Ester Hydrolases; C019921:cocaine esterase; D002091:Butyrylcholinesterase; D003042:Cocaine; D000109:Acetylcholine