An improved cocaine hydrolase: The A328Y mutant of human butyrylcholinesterase is 4-fold more efficient

Weihua Xie, Cibby Varkey Altamirano, Cynthia F. Bartels, Robert J. Speirs, John R. Cashman, Oksana Lockridge

Research output: Contribution to journalArticle

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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 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.

Original languageEnglish (US)
Pages (from-to)83-91
Number of pages9
JournalMolecular pharmacology
Volume55
Issue number1
DOIs
StatePublished - Jan 1 1999

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Butyrylcholinesterase
Hydrolases
Cocaine
Cats
Horses
Benzoylcholine
Chickens
Medical Genetics
Enzymes
Catalytic Domain

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

An improved cocaine hydrolase : The A328Y mutant of human butyrylcholinesterase is 4-fold more efficient. / Xie, Weihua; Altamirano, Cibby Varkey; Bartels, Cynthia F.; Speirs, Robert J.; Cashman, John R.; Lockridge, Oksana.

In: Molecular pharmacology, Vol. 55, No. 1, 01.01.1999, p. 83-91.

Research output: Contribution to journalArticle

Xie, Weihua ; Altamirano, Cibby Varkey ; Bartels, Cynthia F. ; Speirs, Robert J. ; Cashman, John R. ; Lockridge, Oksana. / An improved cocaine hydrolase : The A328Y mutant of human butyrylcholinesterase is 4-fold more efficient. In: Molecular pharmacology. 1999 ; Vol. 55, No. 1. pp. 83-91.
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