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

Abstract

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 [11C](−)-cocaine and [11C](+)-cocaine tracers in human subjects, only [11C](−)-cocaine was observed in the brain, whereas no significant [11C](+)-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 (kcat/KM) against (+)- and (−)-cocaine, and indeed has a catalytic efficiency (kcat/KM=1.84×109M−1min−1) against (−)-cocaine comparable to that (kcat/KM=1.37×109M−1min−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.