On the active site of liver acetyl-CoA. Arylamine N-acetyltransferase from rapid acetylator rabbits (III/J).
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.
D000083:Acetanilides / Q000494:pharmacology; D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000101:Acetyl-CoA C-Acetyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000123:Acetyltransferases / Q000378:metabolism*; D000478:Alkylation; D000596:Amino Acids / Q000032:analysis; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D005838:Genotype; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D008099:Liver / Q000201:enzymology*; D051379:Mice; D008805:Mice, Inbred A / Q000235:genetics; D008810:Mice, Inbred C57BL / Q000235:genetics; D011817:Rabbits
D000083:Acetanilides; D000596:Amino Acids; D000814:Aniline Compounds; D007461:Iodoacetates; C056005:bromoacetanilide; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D000101:Acetyl-CoA C-Acetyltransferase; C056014:4-ethylaniline; D019807:Iodoacetic Acid