A proximal tryptophan in NO synthase controls activity by a novel mechanism
A proximal tryptophan in NO synthase controls activity by a novel mechanism
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DOI：
10.1016/s0162-0134(00)00176-8
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发表时间：
2001-02-01
影响因子：
3.9
通讯作者：
Stuehr, DJ
中科院分区：
文献类型：
--
作者：
Adak, S;Stuehr, DJ
神经元型一氧化氮合酶（nNOS）的血红素参与O-2激活，但也结合自身产生的NO，导致可逆的反馈抑制。我们利用诱变来研究保守的色氨酸残基（Trp 409），其与血红素和氢键结合到其轴向半胱氨酸配体的π堆积，是否有助于控制NO的催化rind调节。突变体W 409 F和W 409 Y在NO合成方面过度活跃，而不影响细胞色素c还原，还原酶独立的N-羟基-精氨酸氧化。或Arg和四氢生物蝶呤结合。在没有精氨酸的情况下，通过血红素的电子通量是慢的W 409突变体比野生型。然而，较少的NO复合物积累过程中NO合成的突变体。为了理解的机制，我们比较了动力学的血红素-NO复合物的形成，血红素还原率，K（猫）之前和之后NO复合物的形成，NO结合亲和力，NO复合物的稳定性，以及它与O-2的反应。在NO合成的初始阶段，血红素-NO复合物的形成在W 409 F和W 409 Y中慢三倍和五倍。这对应于较慢的血红素还原。NO复合物的形成抑制野生型营业额7倍，但减少突变体营业额不到2倍，使突变体更高的稳态活动。NO结合动力学突变体和野生型之间是相似的，虽然突变体也形成了一个417 nm的亚铁-NO复合物。氧化亚铁-NO复合物的突变体比野生型快7倍。我们的结论是，突变多动症主要来自于较慢的血红素还原和更快的氧化血红素-NO复合物的O-2。以这种方式，Trp 409突变通过限制稳态期间亚铁-NO复合物的积累来最小化NO反馈抑制。NOS中W 409的保守性表明，该近端Trp可能调节NO反馈抑制，并且对酶的生理功能很重要。(C)2001 Elsevier Science B. V.保留所有权利。
The heme of neuronal nitric oxide synthase (nNOS) participates in O-2 activation but also binds self-generated NO, resulting in reversible feedback inhibition. We utilized mutagenesis to investigate if a conserved tryptophan residue (Trp409), which engages in pi -stacking with the heme and hydrogen bonds to its axial cysteine ligand, helps control catalysis rind regulation by NO. Mutants W409F and W409Y were hyperactive regarding NO synthesis without affecting cytochrome c reduction, reductase-independent N-hydroxy-arginine oxidation. or Arg and tetrahydrobiopterin binding. In the absence of Arg electron flux through the heme was slower in the W409 mutants than in wild-type. However, less NO complex accumulated during NO synthesis by the mutants. To understand the mechanism, we compared the kinetics of heme-NO complex formation, rate of heme reduction, k(cat) prior to and after NO complex formation, NO binding affinity, NO complex stability, and its reaction with O-2. During the initial phase of NO synthesis, heme-NO complex formation was three and five times slower in W409F and W409Y. which corresponded to a slower heme reduction. NO complex formation inhibited wild-type turnover 7-fold but reduced mutant turnover less than 2-fold, giving mutants higher steady-state activities. NO binding kinetics were similar among mutants and wild type, although mutants also formed a 417 nm ferrous-NO complex. Oxidation of ferrous-NO complex was seven times faster in mutants than in wild type. We conclude that mutant hyperactivity primarily derives from slower heme reduction and faster oxidation of the heme-NO complex by O-2. In this way Trp409 mutations minimize NO feedback inhibition by limiting buildup of the ferrous-NO complex during the steady state. Conservation of W409 among NOS suggests that this proximal Trp may regulate NO feedback inhibition and is important for enzyme physiologic function. (C) 2001 Elsevier Science B.V. All rights reserved.