On the active site of liver acetyl-CoA. Arylamine N-acetyltransferase from rapid acetylator rabbits (III/J).

H. H. Andres, A. J. Klem, L. M. Schopfer, J. K. Harrison, W. W. Weber

Research output: Contribution to journalArticle

60 Scopus citations

Abstract

A covalent, catalytic intermediate of cytosolic liver acetyl coenzyme A: arylamine N-acetyltransferase (EC 2.3.1.5) from rapid acetylator rabbits (III/J) was isolated and chemically characterized. The active site was further studied using two covalent inhibitors, [2-3H]iodoacetic acid and bromoacetanilide. Inhibition experiments with [2-3H]iodoacetic acid at pH 6.9 showed that the incorporation of 0.7 mol of [2-3H]iodoacetic acid/mol of N-acetyltransferase led to rapid, irreversible loss of enzyme activity. Preincubation of the enzyme with acetyl coenzyme A (acetyl-CoA) completely protected against inactivation by [2-3H]iodoacetic acid. After incubating the N-acetyltransferase with [2-3H]acetyl-CoA in the absence of an acceptor amine, an acetyl-cysteinyl-enzyme intermediate was isolated and characterized. Preincubation of N-acetyltransferase with iodoacetic acid prevented the incorporation of the [2-3H]acetyl group into the enzyme. The product analog, bromoacetanilide, caused a rapid irreversible loss of N-acetyltransferase activity. The reaction was pseudo first-order and saturated at high bromoacetanilide concentrations (KI = 0.67 mM; k3 = 1 min-1). Preincubation of the enzyme with acetyl-CoA prevented inactivation by the inhibitor. The acceptor amine 4-ethylaniline did not prevent inhibition. Incorporation of the inhibitor was directly proportional to the loss of activity showing a 1:1 stoichiometry of enzyme to inhibitor. The target amino acid was identified as cysteine by amino acid analysis of inhibitor-treated enzyme.

Original languageEnglish (US)
Pages (from-to)7521-7527
Number of pages7
JournalThe Journal of biological chemistry
Volume263
Issue number16
StatePublished - Jun 5 1988

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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