Fast Kinetic Investigations of Nitric Oxide Synthase

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。一氧化氮是由l -精氨酸合成的一氧化氮合酶（NOS）在生理上产生的。哺乳动物中NOS有三种亚型（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物质，以适应其独特的生理作用。在分子水平上确定NOS异构体的催化和调控机制对于理解NO是如何在生理上产生和管理的，以及设计选择性靶向每个NOS异构体的治疗药物至关重要。我们的长期目标是确定NOS调节和产生NO的分子机制，从而更好地理解这些过程是如何被控制和调节的。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生成的快速催化中间体有哪些？我们的中心假设是，辅因子与NOS的结合诱导了直接影响活性部位的构象变化，其基本原理是理解辅因子和底物结合如何调节血红素反应性的机制对于理解NO如何内源性产生和管理至关重要。在本研究中，我们的目标是：1)确定钙调素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)在催化循环的每个步骤中识别和表征快速中间体。这些目标将通过多通道（200-800 nm）基于激光的纳秒时间分辨光谱、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来实现。由于NOS酶在人类健康和疾病发病机制中发挥着多种作用，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调控的分子机制和对其催化机制的更清晰描述，将促进对NOS在疾病和健康中的作用的认识。了解一氧化氮是如何被调节并阐明其催化机制对于设计控制一氧化氮合成的治疗方法以及了解一氧化氮生理受损如何导致有害的健康影响至关重要。

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.