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 and K(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)/Km). 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 cocane hydrolase activity. The J and K variants (E497V and A539T) had k(cat) and K(m) values similar to wild type, but because these variants are reduced to 66 and 33% of normal levelsin 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.
ASJC Scopus subject areas
- Molecular Medicine