Sennequier N, Stuehr D J
Department of Immunology, Cleveland Clinic, Ohio 44195, USA.
Biochemistry. 1996 May 7;35(18):5883-92. doi: 10.1021/bi952844e.
Inducible nitric oxide synthase (iNOS) catalyzes the NADPH-dependent formation of nitric oxide (NO) and citrulline from L-arginine and O2. In addition to serving as substrate, L-arginine alters the enzyme's heme iron spin equilibrium, increases its NADPH oxidation, and promotes assembly of active dimeric iNOS from inactive monomers. To understand what structural aspects of L-arginine are important for causing these effects, we have studied the interactions of iNOS with several L-arginine and guanidine analogs. Very few analogs supported NO synthesis even when bound to iNOS at saturating or near-saturating levels. In contrast, almost all analogs shifted the heme iron spin equilibrium and either increased or decreased NADPH oxidation by iNOS. The guanidine analogs displayed the same pattern of effects as their amino acid counterparts but exhibited a lower affinity except for analogs containing S-alkylisothiourea or aminoguanidine groups. Most analogs also promoted iNOS dimerization, with hydroxyguanidine and S-ethylisothiourea promoting more dimerization than L-arginine itself. Although the analog concentrations required to promote dimerization of monomers were somewhat higher than those required for binding to dimeric iNOS, they followed the same rank order. The degree of dimerization promoted by each analog did not correlate to its binding affinity, its causing a high- or low-spin shift in heme iron spin state, or to its increasing or decreasing NADPH oxidation. Together, we conclude that the enzyme's high degree of substrate specificity only applies to NO synthesis, in that a number of "inactive" structural analogs still bind to iNOS and affect its heme chemistry and structure in the absence of supporting NO synthesis. These latter affects are mediated through binding of the guanidinium portion of L-arginine and its analogs to a single site within iNOS and are relatively independent of the amino acid portion of the molecule.
诱导型一氧化氮合酶（iNOS）催化L-精氨酸和O₂依赖NADPH生成一氧化氮（NO）和瓜氨酸。除作为底物外，L-精氨酸还会改变该酶的血红素铁自旋平衡，增加其NADPH氧化，并促进活性二聚体iNOS从无活性单体组装形成。为了解L-精氨酸的哪些结构方面对于产生这些效应很重要，我们研究了iNOS与几种L-精氨酸和胍类似物的相互作用。即使以饱和或接近饱和水平与iNOS结合，也只有极少数类似物支持NO合成。相比之下，几乎所有类似物都改变了血红素铁自旋平衡，并增加或降低了iNOS的NADPH氧化。胍类似物表现出与其氨基酸对应物相同的效应模式，但除含有S-烷基异硫脲或氨基胍基团的类似物外，其亲和力较低。大多数类似物还促进iNOS二聚化，其中羟基胍和S-乙基异硫脲比L-精氨酸本身更能促进二聚化。虽然促进单体二聚化所需的类似物浓度略高于与二聚体iNOS结合所需的浓度，但它们遵循相同的排序顺序。每种类似物促进二聚化的程度与其结合亲和力、导致血红素铁自旋状态高自旋或低自旋转变，或增加或降低NADPH氧化均无相关性。我们共同得出结论，该酶的高度底物特异性仅适用于NO合成，因为许多“无活性”结构类似物仍可与iNOS结合，并在不支持NO合成的情况下影响其血红素化学和结构。后一种影响是通过L-精氨酸及其类似物的胍基部分与iNOS内的单个位点结合介导的，并且相对独立于分子的氨基酸部分。
