The mechanisms of nitric oxyde synthesis by nitric oxide synthases

一氧化氮合酶(NOSS)合成信号转导和氧化应激分子一氧化氮(NO)。这一研究计划的长期目标是确定NOSS活性中心的结构、功能和动力学性质，以便在分子水平上了解酶活性的催化循环和调节机制。NOSS合成的NO被用作血管和神经系统的信号分子。它也是一种有毒分子，免疫系统用来防御病原体和癌细胞。NO参与了包括动脉粥样硬化、阿尔茨海默病和帕金森病在内的人类生理和疾病的多个方面，这意味着需要异构体特异性的抑制物来调节神经型一氧化氮合酶(NNOS)、内皮型一氧化氮合酶(ENOS)和诱导型一氧化氮合酶(INOS)。我们预计，对NOSS的催化机制，包括提供血红素-配体底物相互作用的详细图像和确定关键的动力学中间体，将提高我们对NOSS化学的理解，从长远来看，将有助于开发有效的、异构体特异性的人类NOSS抑制剂。因此，这项建议的具体目标是：a-用共振拉曼光谱表征在使用非天然底物的NOSS催化循环中形成的第一个含氧中间体，并表征活性中心突变体的含氧复合体。这些研究将有助于确定底物和活性部位上与血红素结合的氧之间的极性和空间相互作用在催化循环中的重要性，也将有助于破译连接到血红素的近端半胱氨酸所起的作用。B-表征一氧化氮合酶突变体的氧化形式，以确定蛋白质动力学在催化循环中的参与。C-表征与过氧化氢反应形成的催化中间体。后两项研究旨在更好地了解一氧化氮合酶合成一氧化氮合酶的机制。D-表征血红素结合配体(O2、NO和CO)和一氧化氮合酶抑制剂与所有三种哺乳动物NOS之间的相互作用，以破译抑制剂结合和异构体特异性的结构基础。综上所述，这些研究将在分子水平上提供对NO合成机制的理解，确定调控酶活性的重要因素，并确定NOSS异构体特异性的结构决定因素。

Nitric oxide synthases (NOSs) synthesize the signaling and oxidative stress molecule nitric oxide (NO). The long-term objectives of this research program are to determine the structural, functional and dynamic properties of the active site of NOSs to understand the catalytic cycle and mechanisms of regulation of the enzymatic activity at the molecular level. The NO synthesized by NOSs is used as a signaling molecule in the vascular and neural systems. It is also a toxic molecule used by the immune system in host defenses against pathogens and cancer cells. The involvement of NO in many aspects of human physiology and diseases, including atherosclerosis, Alzheimer's and Parkinson's, means that isoform-specific inhibitors are needed to regulate neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). We expect that gaining a thorough understanding of the catalytic mechanisms of NOSs, including providing a detailed picture of heme-ligand substrate interactions and identifying key kinetic intermediates, will improve our comprehension of the chemistry of NOSs and, in the long term, will help in the development of potent, isoform-specific inhibitors of human NOSs. The specific objectives of this proposal thus are: A-Characterize by resonance Raman spectroscopy the first oxygenated intermediate formed during the catalytic cycle of NOSs using non-natural substrates and characterize the oxygenated complex of active site mutants. These studies will help determine the importance in the catalytic cycle of polar and steric interactions between substrates and the heme-bound dioxygen at the active site and will also help decipher the role played by the proximal cysteine ligated to the heme. B- Characterize the oxygenated form of NOS mutants to determine the involvement of protein dynamics in the catalytic cycle. C-Characterize catalytic intermediates formed by the reaction with peroxide. The latter two studies aim to get a better understanding of the mechanisms of NOS synthesis by NOSs. D-Characterize the interactions between heme-bound ligands (O2, NO and CO) and NOS inhibitors with all three mammalian NOSs to decipher the structural basis for inhibitor binding and isoform specificity. Together, these studies will provide an understanding at the molecular level of the mechanism of NO synthesis, identify important factors modulating the enzymatic activity and identify structural determinants for isoform-specificity of NOSs.