An improved cocaine hydrolase: the A328Y mutant of human butyrylcholinesterase is 4-fold more efficient.
Butyrylcholinesterase (BChE) has a major role in cocaine detoxication. The rate at which human BChE hydrolyzes cocaine is slow, with a kcat of 3.9 min(-1) and Km of 14 microM. Our goal was to improve cocaine hydrolase activity by mutating residues near the active site. The mutant A328Y had a kcat of 10.2 min(-1) and Km of 9 microM for a 4-fold improvement in catalytic efficiency (kcat/Km). Since benzoylcholine (kcat 15,000 min(-1)) and cocaine form the same acyl-enzyme intermediate but are hydrolyzed at 4000-fold different rates, it was concluded that a step leading to formation of the acyl-enzyme intermediate was rate-limiting. BChE purified from plasma of cat, horse, and chicken was tested for cocaine hydrolase activity. Compared with human BChE, horse BChE had a 2-fold higher kcat but a lower binding affinity, cat BChE was similar to human, and chicken BChE had only 10% of the catalytic efficiency. Naturally occurring genetic variants of human BChE were tested for cocaine hydrolase activity. The J and K variants (E497V and A539T) had k(cat) and Km values similar to wild-type, but because these variants are reduced to 66 and 33% of normal levels in human blood, respectively, people with these variants may be at risk for cocaine toxicity. The atypical variant (D70G) had a 10-fold lower binding affinity for cocaine, suggesting that persons with the atypical variant of BChE may experience severe or fatal cocaine intoxication when administered a dose of cocaine that is not harmful to others.
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Eppley Institute and Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA.
Altamirano
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Bartels
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Speirs
R J
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Cashman
J R
JR
Lockridge
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D000818:Animals; D001665:Binding Sites; D002091:Butyrylcholinesterase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002645:Chickens; D003042:Cocaine / Q000378:metabolism* / Q000506:poisoning; D062787:Drug Overdose / Q000188:drug therapy; D014644:Genetic Variation; D006736:Horses; D006801:Humans; D006868:Hydrolysis; D009154:Mutation
D002091:Butyrylcholinesterase; D003042:Cocaine