Structure-Function and Reacation Mechanism of eNOS

该提案的总体目标是提供四氢生物蝶呤（BH 4）的功能作用和内皮型一氧化氮合酶（eNOS）的整体反应机制的分子理解。要检验的机制假说是基于还原酶和P450氧化酶之间由钙调蛋白CaM/Ca +2介导的3/2偶联模型。为了检验第一个假说，即BH 4和其自由基中间体之间的有效氧化还原循环保证了NO合酶而不是超氧化物合酶活性，我们将获得完整的动力学数据，不同的生物蝶呤氧化还原物种，包括其自由基中间体在单一和多重转换反应的eNOS。生物蝶呤自由基的动力学将与其他氧化还原中心的氧化还原变化以及底物消失和产物形成相关，以解决加氧酶结构域的完整催化机制。为了解决蛋白质和血红素对生物蝶呤氧化还原行为的影响，将产生参与BH 4结合和血红素反应性的关键残基的位点特异性突变。几种BH 4类似物将用于目的1中描述的动力学实验。生物蝶呤自由基的结构将通过不同的光谱方法和DFT计算来表征。为了验证最后一个假设，即氧化还原状态的黄素和钙调素的结合有决定性的影响的整体周转率和耦合的还原酶和加氧酶的活动，我们将探讨在存在和不存在的钙调素的还原酶结构域的氧化和还原反应机制。我们希望定位限速步骤，即与血红素还原偶联的半反应，定义内部电子转移和构象门控。将通过操纵自抑制序列来评估域通信，并评估CaM对电子转移的影响。第一个目的是解决加氧酶结构域的反应机制和BH 4作为加氧酶反应的两个步骤的电子供体的功能，并在引导NO的形成，而不是硝酰基（HNO）或超氧化物（O2-）的生产。第二个目标将提供有关BH 4和血红素之间的结构/功能关系以及调节BH 4氧化还原行为的因素的关键信息。最后一个目的是了解还原酶和CaM在调控与加氧酶结构域的氧化还原偶联中的作用，从而阐明整个eNOS的反应机制。

The overall goal of this proposal is to provide a molecular understanding of the functional role of tetrahydrobiopterin (BH4) and the overall reaction 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 calmodulin, CaM/Ca +2.To test the first hypothesis that efficient redox cycling between BH4 and its radical intermediate warrants a NO synthase rather than a superoxide synthase activity, we will obtain complete kinetic data of different biopterin redox species including its radical intermediate during single and multiple turnover reaction of eNOS. The kinetics of the biopterin radical will be correlated with redox change of other redox centers and the substrate disappearance and product formation to resolve the full catalytic mechanism of the oxygenase domain. To address the protein and heme effect on the biopterin redox behavior site-specific mutation of critical residues involved in BH4 binding and heme reactivity will be generated. Several BH4 analogs will be utilized in the kinetic experiments described in Aim 1. The structure of the biopterin radical will be characterized by different spectroscopic methods and DFT calculations. To test the last hypotheses that redox state of the flavins and the binding of CaM have deciding effect on the overall turnover rate and coupling of the reductase and oxygenase activities, we will look into the oxidative and reductive reaction mechanism of the reductase domain in the presence and absence of CaM. We wish to locate the rate-limiting step(s), the half-reaction that couples to heme reduction, defining the internal electron transfer and conformational gating. Domain communication will be assessed by manipulating the autoinhibitory sequence and to evaluate the effect of CaM on electron transfer. The first Aim is to address the reaction mechanism of the oxygenase domain and the function of BH4 as the electron donor for the two steps of oxygenase reaction, and in steering NO formation instead of nitroxyl (HNO) or superoxide (O2-) production. The second aim will provide critical information regarding the structure/function relationship between the BH4 and heme and the factors that regulate BH4 redox behaviors. The last Aim will provide understanding of the role of reductase and CaM in regulating the redox coupling with the oxygenase domain thus lead to an elucidation of the reaction of the reaction mechanism of the whole eNOS.