STRUCTURE /FUNCTION AND REACTION MECHANISM OF NITRIC OXI

本提案的总体目标是提供对内皮型一氧化氮合酶(ENOS)结构/功能关系和酶机制的分子理解。有待检验的机制假说是基于CaM/Ca2介导的还原酶和P450氧化酶之间的3/2偶联模型。我们认为，CaM在FMN和血红素中心之间固定特定的方向，而不显著改变它们的物理距离。我们还假设四氢生物蝶呤(H4B)除了其结构作用外，还参与了加氧酶催化的氧化还原步骤。我们进一步认为，L-精氨酸、H4B和血红素位点之间的密切空间关系导致了每个位点上配体结合的相互调节。这些特定的相互作用在三种一氧化氮合酶亚型中被认为是不同的。这些差异，再加上每种一氧化氮合酶亚型中CaM靶标的低相似性，决定了各个亚型中的限速步骤和观察到的周转次数。为了检验这些假设，我们建议：(I)。制备eNOS和具有完整氧化还原中心的两个单独结构域，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)通过结合天然配体和类似物的光谱和动力学方法评估精氨酸、H4B和血红素结合位点之间的相互作用；研究eNOS及其氧合酶和还原酶域的电子传递序列和动力学，并利用快速扫描停流、快速冷冻EPR和快速猝灭/高效液相分析来评估所提出的机制和CaM和H4B的调节作用。计划中的研究将产生关于eNOS(和其他NOS亚型)如何发挥作用的综合知识，并为设计控制与NO相关的病理生理事件的选择性药物抑制剂提供有用的结构信息。

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.