Biochemistry of Nitric Oxide Synthesis
一氧化氮合成的生物化学
基本信息
- 批准号:7422279
- 负责人:
- 金额:$ 27.95万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:1991
- 资助国家:美国
- 起止时间:1991-01-01 至 2011-05-31
- 项目状态:已结题
- 来源:
- 关键词:AnabolismArginineBackBindingBiochemicalBiochemistryBiologicalCatalysisChemicalsClinicalComplicationDiseaseElectron TransportElectron transfer flavoproteinElectronicsElectronsElementsEnvironmentEnzymesFlavinsGoalsGrantHealthHemeHumanHydroxylationInvestigationIsoenzymesKineticsLinkMethodsMolecularMonitorMutationN hydroxylationNitric OxideNitric Oxide SynthaseNitrogenObject AttachmentOxidation-ReductionOxygenProductionProteinsPterinsReactionRegulationRelative (related person)Research PersonnelSiteStructureStructure-Activity RelationshipTestingThermodynamicsThinkingThioamidesWorkanalogbasecofactorheme ahuman diseaseimprovedmutantnoveloxidationprogramstetrahydrobiopterin
项目摘要
DESCRIPTION (provided by applicant): Three NO synthases (iNOS, nNOS, and eNOS) function broadly in human health and disease. Our goal is to define the mechanism of NO synthesis and the structure-function aspects of NOS catalysis, which should help to develop clinical strategies to control NO availability. All NOS contain heme and flavins and catalyze a two-step oxidation of L-arginine to make NO. NOSs are unique among flavoheme enzymes because they contain 6R-tetrahydrobiopterin (HUB) as a bound cofactor. Moreover, their H4B cofactor undergoes novel one- electron transitions during catalysis. Our previous studies showed how H4B radical formation relates to steps in oxygen activation and catalysis in the three NOS, and probed some of the structure-function aspects of H4B redox function. Our current Aims describe biochemical, kinetic, molecular biological, and biophysical studies that will advance our understanding of NOS catalysis by further defining the mechanisms, regulation, and impact of H4B functions. Aim 1. Investigate the structural, thermodynamic, and NOS isozyme-specific basis for regulating H4B radical formation. H4B transfers an electron to a heme-dioxy intermediate in both reactions of NO synthesis (Arg hydroxylation and N-hydroxyArg oxidation). We hypothesize that the kinetics and extent of H4B radical formation are tuned by surrounding protein residues and by the pterin ring structure itself. We will test the function of specific NOS residues and pterin structural analogs, and investigate thermodynamic control of H4B radical formation in NOS. Aim 2. Investigate redox-independent effects of pterins on the reduction, stability, and subsequent reactivity of the FeII02 intermediate in NOS. We will utilize redox-inactive pterins to investigate how bound pterins impact NOS flavoprotein electron transfer to the heme, the stability of the FeIIO2 intermediate, and the further reactivity of NOS heme-oxy species to generate products from Arg and NONA. This work will provide a comprehensive view of how pterin influences NOS catalysis. Aim 3. Investigate the reductive transitions of the H4B radical and how they are regulated. The H4B radical formed in both reactions of NO synthesis must be reduced back to H4B before the enzyme can continue catalysis. How this occurs is unclear. We hypothesize that the H4B radical is reduced by distinct mechanisms in each reaction of NO synthesis. We have developed methods to monitor H4B radical reduction in single catalytic turnover reactions and will examine the mechanistic and regulatory aspects. Aim 4. Initiate 13C, 15N, and HSQC NMR studies of bound H4B in NOS. We hypothesize that the NOS protein creates an electronic environment for H4B that favors its one-electron redox transitions. We will perform NMR studies on 13C- and 15N-enriched H4B bound in NOS or its mutants to test specific hypotheses derived from our protein crystal structures. Relevance: By clarifying how nitric oxide production occurs and is regulated, our work may help to develop treatments for human diseases that involve making too much or too little nitric oxide.
说明书(申请人提供):三种一氧化氮合酶(iNOS、nNOS和eNOS)在人类健康和疾病中具有广泛的功能。我们的目标是明确NO合成的机制和NOS催化的结构-功能方面,这将有助于开发临床策略来控制NO的可获得性。所有的一氧化氮合酶都含有血红素和黄素,并催化L精氨酸的两步氧化生成一氧化氮。NOSS在黄素酶中是独一无二的,因为它们含有6R-四氢生物蝶呤(HUB)作为结合辅因子。此外,它们的H4B辅助因子在催化过程中经历了新颖的单电子跃迁。我们以前的研究显示了H4B自由基的形成与三种NOS中氧活化和催化步骤的关系,并探讨了H4B氧化还原功能的一些结构-功能方面。我们目前的目标是描述生化、动力学、分子生物学和生物物理研究,这些研究将通过进一步定义H4B功能的机制、调节和影响来促进我们对NOS催化的理解。目的1.研究调控H4B自由基形成的结构、热力学和一氧化氮合酶同工酶的特异性基础。在NO合成的两个反应(精氨酸羟化和N-羟基精氨酸氧化)中,H4B将电子转移到血红素-二氧基中间体。我们假设H4B自由基的形成动力学和程度受到周围蛋白质残基和蝶呤素环结构本身的调节。我们将测试特定的一氧化氮合酶残基和蝶呤结构类似物的功能,并研究一氧化氮合酶中H4B自由基形成的热力学控制。目的2.研究蝶呤类化合物对一氧化氮合酶中FeII02中间体的还原、稳定性和随后的反应活性的非氧化还原非还原作用。我们将利用氧化还原失活的蝶呤来研究结合的蝶呤如何影响一氧化氮合酶黄素蛋白向血红素的电子转移,FeIIO2中间体的稳定性,以及一氧化氮合酶血红素氧基物种进一步从Arg和NONA生成产物的反应能力。这项工作将提供一个关于蝶呤如何影响一氧化氮合酶催化的全面观点。目的3.研究H4B自由基的还原转变及其调控机制。在NO合成的两个反应中形成的H4B自由基必须被还原回H4B,酶才能继续催化。目前还不清楚这是如何发生的。我们假设,在NO合成的每个反应中,H4B自由基都被不同的机制还原。我们已经开发了监测单一催化周转反应中H4B自由基还原的方法,并将研究其机理和调节方面。目的4.启动一氧化氮合酶中结合H4B的~(13)C、~(15)N和~(15)HSQC核磁共振研究。我们假设NOS蛋白为H4B创造了一个有利于其单电子氧化还原转变的电子环境。我们将对结合在NOS或其突变体中的富含13C和15N的H4B进行核磁共振研究,以验证从我们的蛋白质晶体结构得出的特定假设。相关性:通过澄清一氧化氮的产生和调控方式,我们的工作可能有助于开发治疗人类疾病的方法,这些疾病涉及产生过多或过少的一氧化氮。
项目成果
期刊论文数量(0)
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DENNIS J STUEHR其他文献
DENNIS J STUEHR的其他文献
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{{ truncateString('DENNIS J STUEHR', 18)}}的其他基金
Defining a pathway for mitochondrial heme trafficking
定义线粒体血红素运输途径
- 批准号:
10733705 - 财政年份:2023
- 资助金额:
$ 27.95万 - 项目类别:
Coordinate control of hemeprotein maturation and function by cell chaperones, heme, and nitric oxide
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- 批准号:
10207671 - 财政年份:2019
- 资助金额:
$ 27.95万 - 项目类别:
Coordinate control of hemeprotein maturation and function by cell chaperones, heme, and nitric oxide
细胞伴侣、血红素和一氧化氮协调控制血红素蛋白的成熟和功能
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10428556 - 财政年份:2019
- 资助金额:
$ 27.95万 - 项目类别:
New mechanism and regulation of intracellular heme delivery in mammals
哺乳动物细胞内血红素输送的新机制和调控
- 批准号:
8241962 - 财政年份:2011
- 资助金额:
$ 27.95万 - 项目类别:
New mechanism and regulation of intracellular heme delivery in mammals
哺乳动物细胞内血红素输送的新机制和调控
- 批准号:
8636030 - 财政年份:2011
- 资助金额:
$ 27.95万 - 项目类别:
New mechanism and regulation of intracellular heme delivery in mammals
哺乳动物细胞内血红素输送的新机制和调控
- 批准号:
8449268 - 财政年份:2011
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New mechanism and regulation of intracellular heme delivery in mammals
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8082579 - 财政年份:2011
- 资助金额:
$ 27.95万 - 项目类别:
2009 Nitric Oxide Gordon Research Conference
2009 年一氧化氮戈登研究会议
- 批准号:
7600784 - 财政年份:2009
- 资助金额:
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Nitric Oxide Synthases As Oxidative And Therapeutic Agents
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7664775 - 财政年份:2008
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Redor Pathways Regulating Nemeprotein Maturation in Asthma
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9232190 - 财政年份:2006
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