Characterization of the inducible nitric oxide synthase oxygenase domain identifies a 49 amino acid segment required for subunit dimerization and tetrahydrobiopterin interaction
Characterization of the inducible nitric oxide synthase oxygenase domain identifies a 49 amino acid segment required for subunit dimerization and tetrahydrobiopterin interaction
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DOI：
10.1021/bi9702290
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发表时间：
1997-09-02
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：
Ghosh, DK;Wu, CQ;Stuehr, DJ
诱导型 NO 合酶的加氧酶结构域（残基 1-498，iNOSox）是该酶的催化中心。其活性形式是同型二聚体，含有血红素和四氢生物蝶呤 (H4生物蝶呤)，并结合 L-精氨酸 [Ghosh, D. K., & Stuehr, D. J. (1995) Biochemistry 34, 801]，为了帮助识别参与辅基和二聚体相互作用的蛋白质残基，我们在大肠杆菌中表达了不含 H4生物蝶呤的 iNOSox。 iNOSox 是 80% 二聚体，但含有低自旋血红素铁，可作为第六配体结合 DTT。 iNOSox 以高亲和力结合 H4 生物蝶呤或 L-精氨酸，从血红素中取代 DTT，并引起与血红素袋闭合一致的光谱变化。充满 H4 生物蝶呤的 iNOSox 可以在 H2O2 支持的反应中催化 N-omega-羟基精氨酸转化为瓜氨酸和 NO。不含 H4 生物蝶呤的 iNOSox 二聚体的有限胰蛋白酶水解在其 N 末端区域 (K117) 的单个位点切割蛋白质。 H4生物蝶呤可以防止裂解，而 L-精氨酸则不能。所得的 40 kDa 蛋白质含有硫醇连接的低自旋血红素，是单体，无催化活性，没有表现出结合 H4 生物蝶呤或 L-精氨酸的能力，并且在提供这些分子时不会二聚化，表明残基 1-117 对于 iNOSox 二聚化和 H4 生物蝶呤/L-精氨酸相互作用很重要。缺失残基 1-114 的缺失突变体是部分二聚体，但在光谱和催化特性方面与 40 kDa 蛋白相同，并且无法响应 L-精氨酸和 H4生物蝶呤，而缺失残基 1-65 的缺失突变体相当于野生型 iNOSox，将重要区域缩小到氨基酸 66-114。该区域保守半胱氨酸 (C109A) 的突变降低了 H4生物蝶呤亲和力，但不影响 iNOSox 二聚体结构、L-精氨酸结合或催化功能。这些结果表明 iNOSox 的残基 66-114 参与生产性 H4 生物蝶呤相互作用和亚基二聚化。 H4生物蝶呤结合似乎可以稳定该区域的蛋白质结构，并通过这样做激活 iNOS 进行 NO 合成。
The oxygenase domain of inducible NO synthase (residues 1-498, iNOSox) is the enzyme's catalytic center. Its active form is a homodimer that contains heme and tetrahydrobiopterin (H4biopterin) and binds L-arginine [Ghosh, D. K., & Stuehr, D. J. (1995) Biochemistry 34, 801], To help identify protein residues involved in prosthetic group and dimeric interaction, we expressed H4biopterin-free iNOSox in Escherichia coli. The iNOSox was 80% dimeric but contained a low-spin heme iron that bound DTT as a sixth ligand. The iNOSox bound H4biopterin or L-arginine with high affinity, which displaced DTT from the heme and caused spectral changes consistent with a closing up of the heme pocket, The H4biopterin-replete iNOSox could catalyze conversion of N-omega-hydroxyarginine to citrulline and NO in a H2O2-supported reaction. Limited trypsinolysis of the H4biopterin-free iNOSox dimer cut the protein at a single site in its N-terminal region (K117). H4biopterin protected against the cleavage whereas L-arginine did not. The resulting 40 kDa protein contained thiol-ligated low-spin heme, was monomeric, catalytically inactive, showed no capacity to bind H4biopterin or L-arginine, and did not dimerize when provided with these molecules, indicating that residues 1-117 were important for iNOSox dimerization and H4biopterin/L-arginine interaction. A deletion mutant missing residues 1-114 was partially dimeric but otherwise identical to the 40 kDa protein regarding its spectral and catalytic properties and inability to respond to L-arginine and H4biopterin, whereas a deletion mutant missing residues 1-65 was equivalent to wildtype iNOSox, narrowing the region of importance to amino acids 66-114. Mutation of a conserved cysteine in this region (C109A) decreased H4biopterin affinity without compromising iNOSox dimeric structure, L-arginine binding, or catalytic function. These results suggest that residues 66-114 of iNOSox are involved in productive H4biopterin interaction and subunit dimerization. H4biopterin binding appears to stabilize the protein structure in this region, and through doing so activates iNOS for NO synthesis.