TY - JOUR T1 - An Improved Cocaine Hydrolase: The A328Y Mutant of Human Butyrylcholinesterase is 4-fold More Efficient JF - Molecular Pharmacology JO - Mol Pharmacol SP - 83 LP - 91 DO - 10.1124/mol.55.1.83 VL - 55 IS - 1 AU - Weihua Xie AU - Cibby Varkey Altamirano AU - Cynthia F. Bartels AU - Robert J. Speirs AU - John R. Cashman AU - Oksana Lockridge Y1 - 1999/01/01 UR - http://molpharm.aspetjournals.org/content/55/1/83.abstract N2 - Butyrylcholinesterase (BChE) has a major role in cocaine detoxication. The rate at which human BChE hydrolyzes cocaine is slow, with ak cat of 3.9 min−1 andK m of 14 μM. Our goal was to improve cocaine hydrolase activity by mutating residues near the active site. The mutant A328Y had a k cat of 10.2 min−1 and K m of 9 μM for a 4-fold improvement in catalytic efficiency (k cat/K m). Since benzoylcholine (k cat 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 k cat 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 catand K m 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. ER -