Fast Kinetic Investigations of Nitric Oxide Synthase

一氧化氮合酶的快速动力学研究

• 批准号: 8634126
• 负责人: Raymond M. Esquerra
• 金额: $ 11.55万
• 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/8634126
• 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）参与了许多生理功能，包括血管舒张，神经传递和免疫系统的细胞毒性作用。 NO是由氨基酸L-精氨酸的酶一氧化氮合酶（NOS）在生理上产生的。哺乳动物（内皮，神经元和诱导）中有三种NOS的同工型，每种NOS都会发展出特定的机制和化学物质，以适合其独特的生理作用。确定分子水平的NOS同工型的催化和调节机制对于了解如何在生理上产生和管理NO，以及设计选择性地靶向每个NOS同工型的治疗剂。我们的长期目标是定义NOS调节和生产NO的分子机制，从而更好地了解这些过程如何受到控制和调节。我们的目标是回答以下问题：1）辅助因子和底物的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）在无生产机理期间，快速催化中间体是什么？我们的中心假设是，辅助因子与NOS的结合诱导了直接影响活性位点的构象变化，理解理解如何通过辅助因子和底物结合调节血红素反应性的机制对于理解如何生产和依据地构成了无生产和管理。在此提案中，我们的目的是：1）确定钙调蛋白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）在催化循环的每个步骤中识别和表征快速中间体。这些目标将使用多通道（200-800 nm）激光基于纳秒时间分辨的光谱与流动式弹性混合结合焦距诱变，以确定辅助因子和底物结合如何影响蜂羊反应性。由于NOS酶在人类健康和疾病发病机理中起多种作用，因此我们希望确定蛋白质基质如何调节活性并阐明催化的机制。 NOS调控的分子机制以及对这项工作将导致的催化机理的更清晰的描述将促进对NOS在疾病和健康中所起的作用的理解。了解如何调节NOS并阐明其催化机制对于设计控制不合成的疗法和了解如何损害生理学会导致有害的健康效应，这至关重要。