STRUCTURE /FUNCTION AND REACTION MECHANISM OF NITRIC OXI

本研究的总体目标是从分子水平上了解内皮型一氧化氮合酶（eNOS）的结构/功能关系和酶的作用机制。待测试的机制假设是基于还原酶和P450氧化酶之间由CaM/Ca+2介导的3/2偶联模型。我们建议，钙调素固定之间的FMN和血红素中心的物理距离没有剧烈变化的特定方向。我们还推测，四氢生物蝶呤（H4 B），除了其结构的作用，是参与加氧酶催化的氧化还原步骤。我们进一步表明，L-精氨酸，H4 B和血红素网站的亲密的空间关系，导致配体结合到每个网站的相互调节。这些特定的相互作用被认为是不同的三种NOS亚型。这种差异，连同在CaM目标在每个NOS亚型的低相似性，确定限速步骤和观察到的营业额在个别亚型。为了验证这些假设，我们建议：（一）。制备eNOS和两个具有完整的氧化还原中心的单独结构域，并使用化学计量和电位滴定来表征每个氧化还原中心的相对和绝对氧化还原电位;（ii）通过光谱和动力学方法与天然配体和类似物的组合来评估L.精氨酸，H4 B和血红素结合位点之间的相互作用;（iii）.表征eNOS及其加氧酶和还原酶结构域中的电子转移序列和动力学，并使用快速扫描停流、快速冷冻EPR和快速淬灭/HPLC分析来监测各个氧化还原中心，评估所提出的机制和CaM和H4 B的调节作用。计划中的研究将产生关于eNOS（和其他NOS亚型）如何起作用的综合知识，并提供用于设计选择性药理学抑制剂以控制与NO相关的病理生理事件的结构信息。

The overall goal of this proposal is to provide a molecular understanding of the structure/function relationships and enzymic mechanism of endothelial-type nitric oxide synthase (eNOS). The mechanistic hypothesis to be tested is based on a 3/2 coupling model between the reductase and the P450 oxidase mediated by CaM/Ca+2. We propose that CaM fixes a specific orientation between the FMN and heme centers without a drastic change in their physical distance. We also hypothesize that tetrahydrobiopterin (H4B), in addition to its structural role, is involved in the redox steps of oxygenase catalysis. We further suggest that the intimate spatial relationship of the L-arginine, H4B and heme sites leads to mutual regulation of ligand binding to each site. These specific interactions are proposed to be different in the three NOS isoforms. Such differences, together with the low similarity in the CaM target in each NOS isoform, determine the rate-limiting steps and the observed turnover numbers in the individual isoforms. To test these hypotheses we propose to: (i). Prepare eNOS and the two individual domains with a full complement of redox centers and use stoichiometric and potentiometric titrations to characterize the relative and absolute redox potential of each redox center; (ii).Evaluate interactions among L.arginine, H4B and heme binding sites by spectroscopic and kinetic methods with combinations of natural ligands and analogs; (iii). Characterize the electron transfer sequence and kinetics in eNOS and its oxygenase and reductase domains, and evaluate the proposed mechanism and the regulatory roles of CaM and H4B using rapid scan stopped-flow, rapid-freezing EPR and rapid-quenching/HPLC analyses to monitor individual redox centers. The planned studies will yield integrated knowledge about how eNOS (and other NOS isoforms) function, and provide structural information useful for the design of selective pharmacological inhibitors to controlling pathophysiological events associated with NO.