Structure-Function and Reacation Mechanism of eNOS

eNOS的结构功能及反应机制

• 批准号: 6829733
• 负责人: AH-LIM TSAI
• 金额: $ 25.14万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6829733
• 关键词: active sites; binding sites; calmodulin; chemical kinetics; cytochrome P450; electron nuclear double resonance spectroscopy; electron transport; enzyme activity; enzyme mechanism; enzyme model; enzyme structure; heme; high performance liquid chromatography; isozymes; nitric oxide synthase; oxidation reduction reaction; potentiometry; protein structure function; site directed mutagenesis; stoichiometry; stop flow technique; tetrahydrobiopterin

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.

该提案的总体目标是提供对四氢生物蝶呤 (BH4) 功能作用和内皮型一氧化氮合酶 (eNOS) 整体反应机制的分子理解。要测试的机制假设基于钙调蛋白 CaM/Ca +2 介导的还原酶和 P450 氧化酶之间的 3/2 耦合模型。为了测试第一个假设，即 BH4 及其自由基中间体之间的有效氧化还原循环保证 NO 合酶而不是超氧化物合酶活性，我们将获得不同生物蝶呤氧化还原物种（包括其自由基中间体）的完整动力学数据。 和 eNOS 的多重周转反应。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联，以解析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 BH4 结合和血红素反应性的关键残基的位点特异性突变。目标 1 中描述的动力学实验中将使用几种 BH4 类似物。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。为了检验黄素的氧化还原状态和 CaM 的结合对还原酶和加氧酶活性的整体周转率和耦合具有决定性影响的最后假设，我们将研究在存在和不存在 CaM 的情况下还原酶结构域的氧化和还原反应机制。我们希望找到限速步骤，即与血红素还原耦合的半反应，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。第一个目标是解决加氧酶结构域的反应机制以及 BH4 作为加氧酶反应两个步骤的电子供体的功能，以及引导 NO 形成而不是硝酰基 (HNO) 或超氧化物 (O2-) 产生的功能。第二个目标将提供有关 BH4 和血红素之间的结构/功能关系以及调节 BH4 氧化还原行为的因素的关键信息。最后一个目标将提供对还原酶和 CaM 在调节氧化还原与加氧酶结构域偶联中的作用的理解，从而阐明整个 eNOS 的反应机制。