Mutagenesis of acidic residues in the oxygenase domain of inducible nitric-oxide synthase identifies a glutamate involved in arginine binding
Mutagenesis of acidic residues in the oxygenase domain of inducible nitric-oxide synthase identifies a glutamate involved in arginine binding
复制标题
DOI：
10.1021/bi970331x
复制
发表时间：
1997-04-29
期刊：
影响因子：
2.9
通讯作者：
Stuehr, DJ
中科院分区：
文献类型：
--
作者：
Gachhui, R;Ghosh, DK;Stuehr, DJ
小鼠细胞因子诱导的一氧化氮合酶(iNOSox，氨基酸1-498)的氧合酶结构域与血红素、四氢生物蝶呤和底物Arg结合，是催化一氧化氮合成和维持酶活性二聚体结构的结构域。为了进一步了解iNOSox的结构和功能，我们对位于iNOSox(氨基酸352-473)区域内的15个保守的酸性残基进行了丙氨酸点突变，这些残基与二氢叶酸还原酶中的蝶呤结合模块序列同源，可能对iNOx亚单位二聚和/或Arg结合起重要作用。5个点突变与野生型相同或几乎相同，10个点突变表现出一系列缺陷，包括低血红素含量(2)，血红素配基不稳定(2)，二聚化缺陷(2)，精氨酸和/或四氢生物蝶呤结合不良(4)，导致四氢生物蝶呤结合缺陷的突变还与其他缺陷有关。相反，两个突变体(E371A和D376A)在Arg结合上表现出独有的缺陷，这些突变体是二聚体，表明iNOSox在大肠杆菌中的二聚化不需要Arg，其中一例(E371A)的Arg结合缺陷是绝对的，通过光谱微扰、放射性配基结合和催化研究进行了评估。我们的结论是，iNOSox这一区域保守的酸性残基的突变可以导致二聚和Arg结合的排他性缺陷。建模考虑预测，E371羧酸盐可能通过与其胍部分相互作用参与Arg结合。
The oxygenase domain of the mouse cytokine-inducible nitric-oxide synthase (iNOSox, amino acids 1-498) binds heme, tetrahydrobiopterin, and the substrate Arg and is the domain responsible for catalyzing nitric oxide synthesis and maintaining the enzyme's active dimeric structure, To further understand iNOSox structure-function, we carried out alanine point mutagenesis on 15 conserved acidic residues located within a region of iNOSox (amino acids 352-473) that shares sequence homology with the pterin-binding module in dihydrofolate reductases and may be important for iNOSox subunit dimerization and/or Arg binding. Five point mutants were identical or nearly identical to wild-type, while 10 exhibited a range of defects that included low heme content (2), heme ligand instability (2), defective dimerization (2), and poor Arg and/or tetrahydrobiopterin binding (4), Mutations that caused defective tetrahydrobiopterin binding were also associated with other defects. In contrast, two mutants (E371A and D376A) exhibited an exclusive defect in Arg binding, These mutants were dimeric, indicating that dimerization of iNOSox in Escherichia coli does not require Arg, In one case (E371A), the defect in Arg binding was absolute, as assessed by spectral perturbation, radioligand binding, and catalytic studies. We conclude that mutagenesis of conserved acidic residues within this region of iNOSox can lead to exclusive defects in dimerization and in Arg binding, Modeling considerations predict that the E371 carboxylate may participate in Arg binding by interacting with its guanidine moiety.