Catalytic activities of a cocaine hydrolase engineered from human butyrylcholinesterase against (+)- and (-)-cocaine

It can be argued that an ideal anti-cocaine medication would be one that accelerates cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e., hydrolysis catalyzed by butyrylcholinesterase (BChE) in plasma. However, wild-type BChE has a low catalytic efficiency against naturally occurring (-)-cocaine. Interestingly, wild-type BChE has a much higher catalytic activity against unnatural (+)-cocaine. According to available positron emission tomography (PET) imaging analysis using [C-11](-)-cocaine and [C-11](+)-cocaine tracers in human subjects, only [C-11](-)-cocaine was observed in the brain, whereas no significant [C-11](+)-cocaine signal was observed in the brain. The available PET data imply that an effective therapeutic enzyme for treatment of cocaine abuse could be an exogenous cocaine-metabolizing enzyme with a catalytic activity against (-)-cocaine comparable to that of wild-type BChE against (+)-cocaine. Our recently designed A199S/F227A/S287G/A328 W/Y332G mutant of human BChE has a considerably improved catalytic efficiency against (-)-cocaine and has been proven active in vivo. In the present study, we have characterized the catalytic activities of wild-type BChE and the A199S/F227A/S287G/A328 W/Y332G mutant against both (+)- and (-)-cocaine at the same time under the same experimental conditions. Based on the obtained kinetic data, the A199S/F227A/S287G/A328 W/Y332G mutant has a similarly high catalytic efficiency (k(cat)/K-M) against (+)- and ()-cocaine, and indeed has a catalytic efficiency (k(cat)/K-M = 1.84 x 10(9) M-1 min(-1)) against (-)-cocaine comparable to that (k(cat)/K-M = 1.37 x 10(9) M-1 min(-1)) of wild-type BChE against (+)cocaine. Thus, the mutant may be used to effectively prevent (-)-cocaine from entering brain and producing physiological effects in the enzyme-based treatment of cocaine abuse. (C) 2012 Elsevier Ireland Ltd. All rights reserved.