Fast Kinetic Investigations of Nitric Oxide Synthase

一氧化氮合酶的快速动力学研究

• 批准号: 7568886
• 负责人: Raymond M. Esquerra
• 金额: $ 19.39万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7568886
• 关键词: Active Sites; Affect; Amino Acids; Area; Atherosclerosis; Binding; Biology; Biomedical Research; Blood Substitutes; Calmodulin; Carbon Monoxide; Catalysis; Chemistry; Complex; Diabetes Mellitus; Electron Transport; Electrons; Elements; Environment; Enzymes; Genetic Recombination; Goals; Health; Heme; Hypertension; Immune system; Indium; Investigation; Kinetics; Knowledge; Lasers; Ligand Binding; Ligands; Mammals; Measures; Methodology; Methods; Molecular; Monitor; NADP; NOS1 protein, human; Neurons; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitrogen; Outcome; Oxidoreductase; Oxygen; Oxygenases; Physiologic pulse; Physiological; Physiological Processes; Physiology; Play; Principal Investigator; Process; Production; Property; Protein Isoforms; Proteins; Public Health; Pulse taking; Reaction; Regulation; Research; Role; Septic Shock; Sickle Cell Anemia; Signaling Molecule; Source; Spectrum Analysis; Students; Testing; Therapeutic; Therapeutic Agents; Time; Vasodilation; Vasodilation disorder; Work; absorption; base; cofactor; cytokine; cytotoxic; design; drug development; insight; mutant; nanosecond; neurotransmission; oxidation; photolysis; tetrahydrobiopterin; ultraviolet

Nitric Oxide (NO) is involved in numerous physiological functions including vasodilatation.neurotransmission, and cytotoxic actions of the immune system. Understanding NO synthesis by nitric oxide synthase (NOS) will aid in drug development (for hypertension, atherosclerosis, diabetes) and therapeutic treatments (sickle cell anemia, blood substitutes, and septic shock) that utilize NO bioactivity. Determining catalytic and regulatory mechanisms of NOS is critical for understanding how NO is produced and managed physiologically, and for designing therapeutic agents that target NOS function. Determining the molecular mechanisms behind the regulation and physiological production of NO by NOS is our research goal. Our objective is determining how the kinetics of CO, NO, and 62 binding to NOS are controlled by conformational changes induced by cofactors and substrate. Our hypothesis is that the binding of substrates and cofactors has a direct effect on the reactivity and accessibility of the active site. Our rationale is that understanding the modulation of ligand binding and heme reactivity by substrate and cofactor binding is crucial for under-standing how NO is produced and managed endogenously. We will use a specialized multichannel (200-800 nm) laser-based nanosecond time-resolved spectrophotometer to measure the fast kinetics of ligand binding, electrontransfer, and oxygen activation involved in NO synthesis as a function of the binding of substrate and cofactors. Our aims are: 1) By measuring CO bimolecular recombination kinetics as a function of cofactor interactions, determine the structural mechanism for the binding of cofactors altering the reactivity of NOS. Our hypothesis is that the binding of cofactors modulates heme reactivity by inducing conformational changes. 2) Determine how NOS controls the binding and release of NO by measuring recombination kinetics as a function of cofactor interactions. Our hypothesis is that binding cofactors causes structural changes, altering the binding kinetics of NO. 3) Determine the structural mechanism behind CaM regulation in neuronal NOS. The PI hypothesizes that control elements in the reductase domain affect the reactivity of the active site. 4) Determine how the binding of cofactors alters reactivity to oxygen and alters electron transfer reactions of NOS. Our hypothesis is that 02 binding and kinetics are influenced by the binding of cofactors. We will examine the kinetics of oxygen binding and the formation of oxygen activated intermediates in neuronal NOS (nNOS) using nanosecond multichannel absorption spectroscopy after flowflash initiation of the reaction with Oz- Relevance to Public Health: Knowledge of the specific molecular mechanisms of how NO is produced and managed physiologically by the binding of substrates and cofactors is crucial to understanding and controlling NO physiology and understanding how compromised NO physiology leads to deleterious health effects.

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性作用。了解一氧化氮合酶 (NOS) 合成 NO 将 协助药物开发（高血压、动脉粥样硬化、糖尿病）和治疗（镰状细胞 利用 NO 生物活性的贫血、血液代用品和感染性休克）。确定催化和调节 NOS 的机制对于理解 NO 的产生和生理管理至关重要，并且对于 设计针对 NOS 功能的治疗药物。确定背后的分子机制 NOS对NO的调节和生理产生是我们的研究目标。我们的目标是确定如何 CO、NO 和 62 与 NOS 结合的动力学受以下因素诱导的构象变化控制： 辅因子和底物。我们的假设是底物和辅因子的结合对 活性位点的反应性和可及性。我们的基本原理是了解配体的调节 底物和辅因子结合的结合和血红素反应性对于理解 NO 的作用至关重要 内生地产生和管理。我们将使用基于专业多通道（200-800 nm）激光的 纳秒时间分辨分光光度计，用于测量配体结合、电子转移、 NO 合成中涉及的氧活化作为底物结合的函数 辅助因子。我们的目标是：1) 通过测量 CO 双分子重组动力学作为辅因子的函数 相互作用，确定改变 NOS 反应性的辅因子结合的结构机制。 我们的假设是辅助因子的结合通过诱导构象来调节血红素反应性 变化。 2) 通过测量重组确定 NOS 如何控制 NO 的结合和释放 作为辅因子相互作用的函数的动力学。我们的假设是结合辅助因子会导致结构 变化，改变 NO 的结合动力学。 3）确定CaM调节背后的结构机制 在神经元 NOS 中。 PI 假设还原酶结构域中的控制元件会影响 活性位点。 4) 确定辅助因子的结合如何改变对氧的反应性并改变电子 NOS的转移反应。我们的假设是 02 结合和动力学受到 02 结合的影响 辅助因子。我们将研究氧结合的动力学和氧活化的形成 Flowflash 后使用纳秒多通道吸收光谱分析神经元 NOS (nNOS) 中的中间体 与 Oz 反应的引发-与公共卫生的相关性：特定分子的知识 NO 通过底物和辅助因子的结合产生和生理管理的机制 对于理解和控制 NO 生理学以及了解 NO 如何受损至关重要 生理学会导致有害的健康影响。