Catalytic mechanism of hamster arylamine N-acetyltransferase 2.
Catalytic mechanism of hamster arylamine N-acetyltransferase 2.
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DOI：
10.1021/bi047564q
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发表时间：
2005-08
期刊：
影响因子：
2.9
通讯作者：
Haiqing Wang;L. Liu;P. E. Hanna;C. Wagner
中科院分区：
文献类型：
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作者：
Haiqing Wang;L. Liu;P. E. Hanna;C. Wagner
芳香胺N-乙酰基转移酶(NAT)催化乙酰基从乙酰辅酶A向伯芳胺、联胺和联胺的转移，在药物、致癌物和其他外来生物的代谢和生物活性中起着非常重要的作用。该反应遵循乒乓-乒乓球-比-比机理。细菌NAT的结构分析表明，Cys-His-Asp催化三联体在所有已知的NAT中都是严格保守的。以前，我们已经通过动力学和同位素效应研究证明，仓鼠NAT2的乙酰化依赖于硫代-咪唑离子对(Cys-S(-)-His-imh(+))，而不是一般的酸碱催化。此外，我们还证实，在乙酰化酶中间体形成后，活性部位咪唑His-107可能在生理pH下被去质子化。在本文中，我们报道了两种乙酰辅酶A(AcCoA)和对硝基苯乙酸酯(PNPA)和四种芳胺底物催化NA2的稳态动力学研究。K(CAT)/K(AcCoA)的pH依赖关系出现两个拐点，分别位于5.32+/-0.13和8.48+/-0.24。PK(A)为5.32，与先前报道的酶乙酰化pK(A)为5.2几乎相同，重申了反应的前半部分是由活性中心的硫代-咪唑离子对催化的。在8.48处的拐点表明NAT2上的一个pH敏感基团参与了AcCoA的结合。由每种芳胺底物和水的对数k(4)和对数k(H)2(O)与pK(A)的相关性构建的Brnsted图在pK(A)范围内呈线性自由能关系，范围从-1.7(H(2)O)到4.67(PABA)，斜率β(Nuc)=0.80+/-0.1。然而，当pK(A)从4.67(PABA)进一步增加到5.32(苯丙氨酸)时，k(4)值下降了2.5倍。PH-k(CAT)/K(PABA)谱分析表明，k(CAT)/K(PABA)的pK(A)为5.52+/-0.14，溶剂动力学同位素效应(SKIE)为2.01+/-0.04。分别测定了4.8+/-0.1、3.1+/-0.1和3.2+/-0.1对苯甲胺、pABglu和PNA的k(CAT)/K(B)的正常溶剂同位素效应。这些观察结果与pK(A)值或=5.5的芳胺以硫醇酯的亲核攻击为主的脱乙酰化机理是一致的。一般的碱基可能是His-107，因为His-107到Gln和Asn的突变体被发现缺乏催化活性。相反，在pH为5.2-7.5的范围内，没有观察到乙酰化酶的pH依赖的水解率增加。在此基础上，提出了NAT2催化芳香胺乙酰化反应的机理。
Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from AcCoA to primary arylamines, hydrazines, and hydrazides and play a very important role in the metabolism and bioactivation of drugs, carcinogens, and other xenobiotics. The reaction follows a ping-pong bi-bi mechanism. Structure analysis of bacterial NATs revealed a Cys-His-Asp catalytic triad that is strictly conserved in all known NATs. Previously, we have demonstrated by kinetic and isotope effect studies that acetylation of the hamster NAT2 is dependent on a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)) and not a general acid-base catalysis. In addition, we established that, after formation of the acetylated enzyme intermediate, the active-site imidazole, His-107, is likely deprotonated at physiological pH. In this paper, we report steady-state kinetic studies of NAT2 with two acetyl donors, acetyl coenzyme A (AcCoA) and p-nitrophenyl acetate (PNPA), and four arylamine substrates. The pH dependence of k(cat)/K(AcCoA) exhibited two inflection points at 5.32 +/- 0.13 and 8.48 +/- 0.24, respectively. The pK(a) at 5.32 is virtually identical with the previously reported pK(a) of 5.2 for enzyme acetylation, reaffirming that the first half of the reaction is catalyzed by a thiolate-imidazolium ion pair in the active site. The inflection point at 8.48 indicates that a pH-sensitive group on NAT2 is involved in AcCoA binding. A Brønsted plot constructed by the correlation of log k(4) and log k(H)2(O) with the pK(a) for each arylamine substrate and water displays a linear free-energy relationship in the pK(a) range from -1.7 (H(2)O) to 4.67 (PABA), with a slope of beta(nuc) = 0.80 +/- 0.1. However, a further increase of the pK(a) from 4.67 (PABA) to 5.32 (anisidine) resulted in a 2.5-fold decrease in the k(4) value. Analysis of the pH-k(cat)/K(PABA) profile revealed a pK(a) of 5.52 +/- 0.14 and a solvent kinetic isotope effect (SKIE) of 2.01 +/- 0.04 on k(cat)/K(PABA). Normal solvent isotope effects of 4.8 +/- 0.1, 3.1 +/- 0.1, and 3.2 +/- 0.1 on the k(cat)/K(b) for anisidine, pABglu, and PNA, respectively, were also determined. These observations are consistent with a deacetylation mechanism dominated by nucleophilic attack of the thiol ester for arylamines with pK(a) values or=5.5. The general base is likely His-107 because the His-107 to Gln and Asn mutants were found to be devoid of catalytic activity. In contrast, an increase in pH-dependent hydrolysis of the acetylated enzyme was not observed over a pH range of 5.2-7.5. On the basis of these observations, a catalytic mechanism for the acetylation of arylamines by NAT2 is proposed.