STRUCTURE /FUNCTION AND REACTION MECHANISM OF NITRIC OXI

本提案的总体目标是提供内皮型一氧化氮合酶（eNOS）的结构/功能关系和酶学机制的分子理解。要验证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CaM在FMN和血红素中心之间固定一个特定的方向，而不会剧烈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和血红素位点的密切空间关系导致配体与每个位点的结合相互调节。这些特定的相互作用在三种NOS同工异构体中是不同的。这些差异，加上每个NOS同工异构体中CaM目标的低相似性，决定了限速步骤和单个同工异构体中观察到的周转率。为了验证这些假设，我们建议：(1)。制备具有完整氧化还原中心的eNOS和两个单独的结构域，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；(二)。利用光谱和动力学方法评价天然配体和类似物组合下l .精氨酸、H4B和血红素结合位点之间的相互作用；(3)。表征eNOS及其加氧酶和还原酶结构域的电子转移序列和动力学，并利用快速扫描停止流、快速冷冻EPR和快速淬火/HPLC分析来监测单个氧化还原中心，评估CaM和H4B的可能机制和调控作用。计划中的研究将产生关于eNOS（和其他NOS亚型）功能的综合知识，并为设计选择性药理抑制剂以控制与NO相关的病理生理事件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.