Andres H H, Klem A J, Schopfer L M, Harrison J K, Weber W W
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109-0010.
J Biol Chem. 1988 Jun 5;263(16):7521-7.
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.
从快速乙酰化兔（III/J）中分离并对胞质肝乙酰辅酶A：芳胺N-乙酰基转移酶（EC 2.3.1.5）的共价催化中间体进行了化学表征。使用两种共价抑制剂[2-³H]碘乙酸和溴乙酰苯胺对活性位点进行了进一步研究。在pH 6.9条件下用[2-³H]碘乙酸进行的抑制实验表明，每摩尔N-乙酰基转移酶掺入0.7摩尔[2-³H]碘乙酸会导致酶活性迅速、不可逆地丧失。酶与乙酰辅酶A（乙酰-CoA）预孵育可完全保护其免受[2-³H]碘乙酸的失活作用。在没有受体胺的情况下，将N-乙酰基转移酶与[2-³H]乙酰-CoA孵育后，分离并表征了乙酰半胱氨酰-酶中间体。N-乙酰基转移酶与碘乙酸预孵育可阻止[2-³H]乙酰基掺入酶中。产物类似物溴乙酰苯胺导致N-乙酰基转移酶活性迅速不可逆丧失。该反应为准一级反应，在高溴乙酰苯胺浓度下达到饱和（KI = 0.67 mM；k3 = 1分钟⁻¹）。酶与乙酰-CoA预孵育可防止抑制剂使其失活。受体胺4-乙苯胺不能阻止抑制作用。抑制剂的掺入与活性丧失直接成比例，显示酶与抑制剂的化学计量比为1:1。通过对抑制剂处理的酶进行氨基酸分析，确定靶氨基酸为半胱氨酸。
