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 [(11)C](-)-cocaine and [(11)C](+)-cocaine tracers in human subjects, only [(11)C](-)-cocaine was observed in the brain, whereas no significant [(11)C](+)-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 (k(cat/)K(M) = 1.84 × 10(9) M(-1) min(-1)) against (-)-cocaine comparable to that (k(cat)/K(M) = 1.37 × 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.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 789 South Limestone Street, Lexington, KY 40536, USA.
Liu
L
Xue
Shurong
S
Hou
Wenchao
W
Yang
Lei
L
Fang
Fang
F
Zheng
Chang-Guo
CG
Zhan
D001921:Brain / Q000000981:diagnostic imaging / Q000378:metabolism; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002250:Carbon Radioisotopes; D003042:Cocaine / Q000737:chemistry / Q000378:metabolism*; D019970:Cocaine-Related Disorders / Q000000981:diagnostic imaging / Q000188:drug therapy / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049268:Positron-Emission Tomography; D011487:Protein Conformation; D015202:Protein Engineering; D019275:Radiopharmaceuticals; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D002250:Carbon Radioisotopes; D050505:Mutant Proteins; D019275:Radiopharmaceuticals; D011994:Recombinant Proteins; D002091:Butyrylcholinesterase; D003042:Cocaine