Fast Kinetic Investigations of Nitric Oxide Synthase

一氧化氮(NO)参与多种生理功能，包括血管扩张、神经传递、 以及免疫系统的细胞毒作用。了解一氧化氮合酶(NOS)合成NO将 协助药物开发(用于高血压、动脉粥样硬化、糖尿病)和治疗(镰状细胞 贫血、血液替代品和败血症休克)，这些都不具有生物活性。测定催化和调节作用 一氧化氮合酶的机制对于理解一氧化氮是如何在生理上产生和管理的以及对 设计针对一氧化氮合酶功能的治疗剂。确定其背后的分子机制 一氧化氮合酶对NO的调节和生理产生是我们的研究目标。我们的目标是决定如何 一氧化碳、一氧化氮和62与一氧化氮合酶结合的动力学受 辅因子和底物。我们的假设是底物和辅因子的结合对 活动站点的反应性和可访问性。我们的理论基础是理解配体的调制 底物和辅因子结合的结合和血红素的反应性是理解NO的关键 内生性的生产和管理。我们将使用专门的多通道(200-800 nm)激光 纳秒时间分辨分光光度计测量配基结合，电子转移， 以及参与NO合成的氧的激活作为底物和 辅因。我们的目标是：1)通过测量CO双分子复合动力学作为辅因子的函数 相互作用，决定了改变一氧化氮合酶活性的辅因子结合的结构机制。 我们的假设是，辅因子的结合通过诱导构象来调节血红素的反应性 改变。2)通过测量重组来确定一氧化氮合酶如何控制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，结合的辅因子导致结构性 变化，改变了NO.3的结合动力学)决定了CaM调节背后的结构机制 在神经元型一氧化氮合酶。PI假设还原酶域中的控制元件影响酶的反应活性 活动站点。4)确定辅因子的结合如何改变对氧的反应性和改变电子 一氧化氮合酶的转移反应。我们的假设是02的结合和动力学受结合的影响 辅因。我们将研究氧结合的动力学和氧活化的形成。 流动闪光后纳秒多道吸收光谱分析神经元型一氧化氮合酶中间体 与公众健康相关的臭氧反应的启动：特定分子的知识 NO如何通过底物和辅因子的结合产生和生理管理的机制 对于理解和控制NO生理以及了解NO是如何妥协的至关重要 生理学会导致有害的健康影响。

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.