Fast Kinetic Investigations of Nitric Oxide Synthase

一氧化氮合酶的快速动力学研究

• 批准号: 8414792
• 负责人: Raymond M. Esquerra
• 金额: $ 9.71万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-10 至 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8414792
• 关键词: Active Sites; Affect; Amino Acids; Arginine; Atherosclerosis; Award; Binding; Biochemistry; Biomedical Research; Blood Substitutes; Calmodulin; Catalysis; Cations; Cell physiology; Chemistry; Complex; Diabetes Mellitus; Disease; Electron Transport; Elements; Enzymes; Goals; Health; Heme; Human; Hydrogen Peroxide; Hypertension; Immune system; Indium; Investigation; Kinetics; Lasers; Ligand Binding; Mammals; Measures; Molecular; Mutagenesis; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Outcome; Oxidoreductase; Oxygen; Oxygenases; Pathogenesis; Physiological; Physiological Processes; Physiology; Play; Population; Process; Production; Productivity; Protein Dynamics; Protein Isoforms; Proteins; Reaction; Regulation; Research; Research Infrastructure; Role; San Francisco; Septic Shock; Sickle Cell Anemia; Signaling Molecule; Site; Spectrum Analysis; Students; Therapeutic; Therapeutic Agents; Time; Universities; Vasodilation; Work; absorption; base; cofactor; cytotoxic; design; drug development; human NOS3 protein; mutant; nanosecond; neurotransmission; novel; public health relevance; therapy design

DESCRIPTION (provided by applicant): Nitric oxide (NO) is involved in numerous physiological functions, including vasodilatation, neurotransmission, and cytotoxic actions of the immune system. NO is produced physiologically by the enzyme nitric oxide synthase (NOS) from the amino acid L-arginine. There are three isoforms of NOS in mammals (endothelial, neuronal, and inducible), each one evolving specific mechanisms and chemistries to suit their unique physiological roles. Determining the catalytic and regulatory mechanisms of NOS isoforms at the molecular level is critical for understanding how NO is produced and managed physiologically, and for designing therapeutic agents that selectively target each NOS isoform. Our long-term goal is to define the molecular mechanisms behind the regulation and production of NO by NOS, providing a better understanding of how these processes are controlled and regulated. Our objective is to answer the following questions: 1) How do conformational changes induced by the binding of cofactors and substrate influence the reactivity of the heme active site? 2) What are the fast catalytic intermediates during the mechanism of NO production? Our central hypothesis is that the binding of cofactors to NOS induces conformational changes that directly affect the active site, with the rationale that understanding the mechanisms for how heme reactivity is modulated by cofactor and substrate binding is crucial for understanding how NO is produced and managed endogenously. In this proposal, we aim to: 1) Determine the mechanism of how the binding of calmodulin alters the reactivity of the heme active site in neuronal NOS. 2) Identify and characterize fast intermediates during each step of the catalytic cycle. These aims will be accomplished using multi-channel (200-800 nm) laser-based nanosecond time-resolved spectroscopy with flow-flash mixing combined with focused mutagenesis to determine how cofactor and substrate binding influences heme reactivity. Since NOS enzymes play diverse roles in human health and disease pathogenesis, we desire to determine how the protein matrix regulates activity and to clarify the mechanism of catalysis. The molecular mechanism of NOS regulation and the clearer description of the mechanism of catalysis that will result from this work will advance the understanding of the role that NOS plays in disease and health. Understanding how NOS is regulated and clarifying its catalytic mechanism are crucial both for designing therapies that control NO synthesis and for understanding how compromised NO physiology leads to deleterious health effects.

描述（由申请人提供）：一氧化氮（NO）参与多种生理功能，包括血管舒张、神经传递和免疫系统的细胞毒作用。 NO 是由一氧化氮合酶 (NOS) 从氨基酸 L-精氨酸中产生的。哺乳动物中有三种 NOS 亚型（内皮型、神经元型和诱导型），每种亚型都进化出特定的机制和化学作用，以适应其独特的生理作用。在分子水平上确定 NOS 异构体的催化和调节机制对于了解 NO 的生理产生和管理方式以及设计选择性针对每种 NOS 异构体的治疗药物至关重要。我们的长期目标是明确 NOS 调节和产生 NO 背后的分子机制，从而更好地理解这些过程是如何控制和调节的。我们的目标是回答以下问题：1）辅因子和底物结合引起的构象变化如何影响血红素活性位点的反应性？ 2）NO产生过程中的快速催化中间体有哪些？我们的中心假设是辅因子与 NOS 的结合会引起构象变化，从而直接影响活性位点，其基本原理是了解辅因子和底物结合如何调节血红素反应性的机制对于了解 NO 是如何内源性产生和管理的至关重要。在本提案中，我们的目标是： 1) 确定钙调蛋白的结合如何改变神经元 NOS 中血红素活性位点的反应性的机制。 2) 识别并表征催化循环每个步骤中的快速中间体。这些目标将通过使用基于多通道（200-800 nm）激光的纳秒时间分辨光谱、流动闪光混合以及聚焦诱变来确定辅助因子和底物结合如何影响血红素反应性来实现。由于 NOS 酶在人类健康和疾病发病机制中发挥着不同的作用，我们希望确定蛋白质基质如何调节活性并阐明催化机制。这项工作将带来 NOS 调节的分子机制以及对催化机制的更清晰的描述，将促进人们对 NOS 在疾病和健康中所发挥的作用的理解。了解 NOS 的调节方式并阐明其催化机制对于设计控制 NO 合成的疗法以及了解 NO 生理学受损如何导致有害健康影响至关重要。