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Vol. 55, Issue 1, 83-91, January 1999
Eppley Institute and Department of Biochemistry and Molecular
Biology, University of Nebraska Medical Center, Omaha, Nebraska
(W.X., C.V.A., O.L.) and
Human BioMolecular Research Institute,
Seattle, Washington (R.J.S, J.R.C.)
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 µM. Our goal was to improve
cocaine hydrolase activity by mutating residues near the active site.
The mutant A328Y had a kcat of 10.2 min1 and Km of 9 µM for a
4-fold improvement in catalytic efficiency (kcat/Km). Since
benzoylcholine (kcat 15,000 min1) 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 kcat
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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