title: Characterization of a high-activity mutant of human butyrylcholinesterase against (-)-cocaine.
abstract: Cocaine addiction and overdose are a well-known public health problem. There is no approved medication available for cocaine abuse treatment. Our recently designed and discovered high-activity mutant (A199S/S287G/A328W/Y332G) of human butyrylcholinesterase (BChE) has been recognized to be worth exploring for clinical application in humans as a potential anti-cocaine medication. The catalytic rate constant (k(cat)) and Michaelis-Menten constant (K(M)) for (-)-cocaine hydrolysis catalyzed by A199S/S287G/A328W/Y332G BChE (without fusion with any other peptide) have been determined to be 3,060 min(-1) and 3.1 microM, respectively, in the present study. The determined kinetic parameters reveal that the un-fused A199S/S287G/A328W/Y332G mutant has a approximately 1,080-fold improved catalytic efficiency (k(cat)/K(M)) against (-)-cocaine compared to the wild-type BChE. The approximately 1,080-fold improvement in the catalytic efficiency of the un-fused A199S/S287G/A328W/Y332G mutant is very close to the previously reported the approximately 1,000-fold improvement in the catalytic efficiency of the A199S/S287G/A328W/Y332G mutant fused with human serum albumin. These results suggest that the albumin fusion did not significantly change the catalytic efficiency of the BChE mutant while extending the plasma half-life. In addition, we have also examined the catalytic activities of the A199S/S287G/A328W/Y332G mutant against two other substrates, acetylthiocholine (ATC) and butyrylthiocholine (BTC). It has been shown that the A199S/S287G/A328W/Y332G mutations actually decreased the catalytic efficiencies of BChE against ATC and BTC, while considerably improving the catalytic efficiency of BChE against (-)-cocaine.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 725 Rose Street, Lexington, KY 40536, USA.
authors.forename: Wenchao
authors.initials: W
authors.lastname: Yang
authors.forename: Liu
authors.initials: L
authors.lastname: Xue
authors.forename: Lei
authors.initials: L
authors.lastname: Fang
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.forename: Chang-Guo
authors.initials: CG
authors.lastname: Zhan
mesh_terms: D000818:Animals; D055162:Biocatalysis; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003042:Cocaine / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D050505:Mutant Proteins; D002091:Butyrylcholinesterase; D003042:Cocaine