Fast Kinetic Investigations of Nitric Oxide Synthase

一氧化氮合酶的快速动力学研究

• 批准号: 9023559
• 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 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9023559
• 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; NOS3 gene; 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; ethnic diversity; mutant; nanosecond; neurotransmission; novel; 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由一氧化氮合酶（NOS）从氨基酸L-精氨酸生理性地产生。在哺乳动物中有三种NOS亚型（内皮型、神经型和诱导型），每种亚型都进化出特定的机制和化学物质以适应其独特的生理作用。在分子水平上确定NOS亚型的催化和调节机制对于理解NO是如何产生和生理管理的以及设计选择性靶向每个NOS亚型的治疗剂是至关重要的。我们的长期目标是确定NOS调节和产生NO背后的分子机制，更好地了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）由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生成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的结合诱导构象变化，直接影响活性位点，理解血红素反应性如何通过辅因子和底物结合调节的机制对于理解NO是如何产生和内源性管理至关重要。本研究的目的是：1）确定钙调素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)识别和表征催化循环每个步骤中的快速中间体。这些目标将使用多通道（200-800 nm）基于激光的纳秒时间分辨光谱与流动闪光混合结合聚焦诱变来确定辅因子和底物结合如何影响血红素反应性。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。NOS调节的分子机制以及这项工作对催化机制的更清晰描述将促进对NOS在疾病和健康中所起作用的理解。了解NOS是如何调节和澄清其催化机制是至关重要的设计控制NO合成的治疗和了解如何受损NO生理导致有害的健康影响。