Abstract
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.
Footnotes
- Received April 1, 1998.
- Accepted September 15, 1998.
-
Send reprint requests to: Dr. Oksana Lockridge, University of Nebraska Medical Center, Eppley Institute, 600 S. 42nd St., Omaha, NE 68198-6805. E-mail: olockrid{at}mail.unmc.edu
-
Supported by the Nebraska Affiliate of the American Heart Association Grant 9707841S (to O.L.), AASERT Award DAAG55-07-1-0244 from the U.S. Army Research Office (to O.L.), Minority Student and Sciences Teacher Training Program Grant R25RR10280 from the National Center for Research Resources, National Cancer Institute Grant P30 CA36727 to the Eppley Institute, National Institutes of Health Grants DA08531 and DA00269 (to J.R.C.) and DA011707 (to O.L.), and by U.S. Army Medical Research and Materiel Command Grant DAMD17-97-1-7349 (to O.L.). The opinions or assertions contained herein belong to the authors and should not be construed as the official views of the U.S. Army or the Department of Defense.
- The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|