Cheon H G, Boteju L W, Hanna P E
Department of Pharmacology, University of Minnesota, Minneapolis 55455.
Mol Pharmacol. 1992 Jul;42(1):82-93.
N-Acetyltransferases (NATs) play key roles in the detoxification and/or bioactivation of arylamines, arylhydroxylamines, arylhydroxamic acids, and hydrazines in mammalian tissues. In the present study, two hamster hepatic NATs (NAT I and NAT II) were separated, and each was purified greater than 2000-fold by sequential ammonium sulfate fractionation, DEAE anion exchange chromatography, Sephadex G-75 gel filtration chromatography, aminoazobenzene-coupled affinity chromatography, and DEAE anion exchange high performance liquid chromatography. Both NAT I and NAT II were purified to near-homogeneity. The molecular masses of NAT I and NAT II were estimated to be 30.5 kDa and 32.6 kDa, respectively. 2-(Bromoacetylamino)fluorene (Br-AAF) and bromoacetanilide were synthesized and evaluated as affinity labels for NAT I and NAT II. Whereas Br-AAF was a highly selective inactivator of NAT II, bromoacetanilide inactivated both NAT I and NAT II in a similar fashion. Inactivation of NAT II by both Br-AAF and bromoacetanilide, and inactivation of NAT I by bromoacetanilide, followed pseudo-first-order kinetics. Relative rate constants (k(obs)/[I]) for the two compounds indicate that Br-AAF is approximately 25 times more potent than bromoacetanilide as an inactivator of NAT II. Both acetylcoenzyme A (CoASAc) and 2-acetylaminofluorene protected NAT II from inactivation by Br-AAF, and CoASAc provided protection of both NAT I and NAT II activities from inactivation by bromoacetanilide, indicating that the inactivation by both bromoacetanilide and Br-AAF is active site directed. The irreversibility of the inactivation of NATs by Br-AAF and bromoacetanilide was demonstrated by the failure to recover transacetylase activities after gel filtration of enzyme preparations that had been preincubated with Br-AAF or bromoacetanilide. Preincubation of NAT II with CoASAc significantly reduced the incorporation of [14C]Br-AAF into the enzyme, providing further evidence that the labeling is active site directed. In addition, pretreatment of NAT II with N-ethylmaleimide completely prevented the labeling of NAT II with [14C]Br-AAF, which suggests that a cysteine thiol is the target nucleophile of Br-AAF. High performance liquid chromatography analysis of the hydrochloric acid hydrolysate of [14C]Br-AAF-labeled NAT II revealed that 70% of total radioactivity is associated with S-carboxymethyl-L-cysteine, indicating that Br-AAF reacts primarily with a cysteine residue at the active site. These studies provide direct evidence that hamster hepatic NAT II contains an essential cysteine residue at the active site, and they establish the potential utility of Br-AAF for determining amino acid sequences in the active site of hamster hepatic NAT II.
N - 乙酰基转移酶（NATs）在哺乳动物组织中对芳胺、芳羟胺、芳基异羟肟酸和肼的解毒和/或生物活化过程中发挥关键作用。在本研究中，分离出了两种仓鼠肝脏NATs（NAT I和NAT II），并通过硫酸铵分级沉淀、DEAE阴离子交换色谱、Sephadex G - 75凝胶过滤色谱、氨基偶氮苯偶联亲和色谱以及DEAE阴离子交换高效液相色谱依次进行纯化，每种酶的纯化倍数均超过2000倍。NAT I和NAT II均被纯化至接近均一状态。NAT I和NAT II的分子量估计分别为30.5 kDa和32.6 kDa。合成了2 - （溴乙酰氨基）芴（Br - AAF）和溴乙酰苯胺，并将其作为NAT I和NAT II的亲和标记物进行评估。虽然Br - AAF是NAT II的高度选择性失活剂，但溴乙酰苯胺以类似方式使NAT I和NAT II均失活。Br - AAF和溴乙酰苯胺对NAT II的失活以及溴乙酰苯胺对NAT I的失活均遵循假一级动力学。两种化合物的相对速率常数（k(obs)/[I]）表明，作为NAT II的失活剂，Br - AAF的效力约为溴乙酰苯胺的25倍。乙酰辅酶A（CoASAc）和2 - 乙酰氨基芴均可保护NAT II不被Br - AAF失活，并且CoASAc可保护NAT I和NAT II的活性不被溴乙酰苯胺失活，这表明溴乙酰苯胺和Br - AAF的失活作用均是活性位点导向的。在用Br - AAF或溴乙酰苯胺预孵育的酶制剂经凝胶过滤后未能恢复转乙酰酶活性，这证明了Br - AAF和溴乙酰苯胺对NATs的失活作用是不可逆的。用CoASAc对NAT II进行预孵育可显著减少[14C]Br - AAF掺入酶中，这进一步证明标记是活性位点导向的。此外，用N - 乙基马来酰亚胺对NAT II进行预处理可完全阻止[14C]Br - AAF对NAT II的标记，这表明半胱氨酸硫醇是Br - AAF的靶亲核基团。对[14C]Br - AAF标记的NAT II的盐酸水解产物进行高效液相色谱分析显示，总放射性的70%与S - 羧甲基 - L - 半胱氨酸相关，这表明Br - AAF主要与活性位点的半胱氨酸残基反应。这些研究提供了直接证据，证明仓鼠肝脏NAT II在活性位点含有一个必需的半胱氨酸残基，并确立了Br - AAF在确定仓鼠肝脏NAT II活性位点氨基酸序列方面的潜在用途。
