Identification of amino acids imparting acceptor substrate selectivity to human arylamine acetyltransferases NAT1 and NAT2
Identification of amino acids imparting acceptor substrate selectivity to human arylamine acetyltransferases NAT1 and NAT2
复制标题
DOI：
10.1042/0264-6021:3480159
复制
发表时间：
2000-05-15
影响因子：
4.1
通讯作者：
Grant, DM
中科院分区：
文献类型：
--
作者：
Goodfellow, GH;Dupret, JM;Grant, DM
人芳胺N-乙酰转移酶NAT1和NAT2催化原芳胺和肼的N-乙酰化和o -乙酰化及其n羟基化代谢物。我们之前使用了一组重组NAT1 /NAT2嵌合蛋白来鉴定线性氨基酸片段，这些氨基酸片段在赋予这些蛋白质不同的催化特异性方面发挥作用[Dupret， Goodfellow， Janezic和Grant (1994) J. Biol]。化学学报，26(2):387 - 398。这些研究表明，与含有活性位点半胱氨酸残基Cys（68）不同的保守中心区域（残基112-210）在决定NAT底物选择性方面很重要。在目前的研究中，我们通过进一步的嵌合体产生这个保守区域和随后的单个氨基酸的定点突变来改进我们的分析。对这些突变蛋白与nat1和nat2选择性底物对氨基水杨酸（PAS）和磺胺嘧啶（SMZ）的酶动力学分析表明，残基125、127和129是nat1型和nat2型底物选择性的重要决定因素。Arg（127）的修饰对PAS特异性的影响最大，而改变Phe（125）对SMZ特异性的影响最大。选择的NAT突变体显示乙酰辅酶a的K-m值与野生型NAT相当，这意味着突变影响受体底物特异性而不是辅因子结合亲和力。结合先前的观察结果，这些结果表明，残基125、127和129可能有助于形成围绕Cys的活性位点口袋（68），并作为NAT底物选择性的重要决定因素。
The human arylamine N-acetyltransferases NAT1 and NAT2 catalyse the acetyl-CoA-dependent N- and O-acetylation of primary arylamine and hydrazine xenobiotics and their Nhydroxylated metabolites. We previously used a panel of recombinant NAT1 /NAT2 chimaeric proteins to identify linear amino acid segments that have roles in imparting the distinct catalytic specificities to these proteins [Dupret, Goodfellow, Janezic and Grant (1994) J. Biol. Chem. 269, 26830-26835]. These studies indicated that a conserved central region (residues 112-210) distinct from that containing the active-site cysteine residue Cys(68) was important in determining NAT substrate selectivity. In the present study we have refined our analysis through further chimaera generation of this conserved region and by subsequent site-directed mutagenesis of individual amino acids. Enzyme kinetic analysis of these mutant proteins with the NAT1-selective and NAT2-selective substrates p-aminosalicylic acid (PAS) and sulphamethazine (SMZ) respectively suggests that residues 125, 127 and 129 are important determinants of NAT1-type and NAT2-type substrate selectivity. Modification of Arg(127) had the greatest effect on specificity for PAS, whereas changing Phe(125) had the greatest effect on specificity for SMZ. Selected NAT mutants exhibited K-m values for acetyl-CoA that were comparable with those of the wild-type NATs, implying that the mutations affected acceptor substrate specificity rather than cofactor binding affinity. Taken together with previous observations, these results suggest that residues 125, 127 and 129 might contribute to the formation of the active-site pocket surrounding Cys(68) and function as important determinants of NAT substrate selectivity.