STRUCTURAL METALLOBIOCHEMISTRY OF NITRIC OXIDE SYNTHASES

描述：金属酶一氧化氮合酶(NOS)调节一氧化氮 氧化物(NO)的合成及其生物活性。NO有双重身份 作用：1)神经传递的可扩散生物信使， 长时程增强、血小板聚集和血压调节 2)抗肿瘤细胞和细胞内防御的细胞毒剂 寄生虫。一氧化氮合酶(NOS)，存在于诱导型(INOS)中，构成 内皮细胞(ENOS)和构成神经元(NNOS)亚型，实现其 通过采用耐人寻味的钙调节来实现重要的生物学功能 催化机制，并结合了五个辅因子的独特组装： 血红素、四氢生物蝶呤(H4B)、FMN、FAD和NADPH。NOSS生成无依据 在两步机制的每一步中消耗分子氧和NADPH： 首先，一个类似单加氧酶的反应将L精氨酸转化为中间体 N4-羟基-L-精氨酸(Noh-Arg)，然后一个史无前例的反应转化 能精制成瓜氨酸，不能。以原子细节理解独一无二的 NOSS的结构矿物生物化学、集成结晶学和 建议对三类主要的一氧化氮合酶进行生化研究。 每个一氧化氮合酶亚基被分成两个结构域，由一个钙调蛋白结合连接 铰链区：一个与血红素、H4B和L-精氨酸结合的加氧酶结构域 形成NO产生的催化中心的位置和还原酶结构域 NADPH、FAD和FMN结合部位向血红素提供电子。 从黄素到血红素的电子转移受钙调蛋白控制 (CaM)，它为钙结合蛋白发挥了一个新的作用。这个 拟议的研究首先旨在表征结构生物化学 单独的加氧酶和还原酶结构域，已经过度表达 并结晶，然后使用这些结果来确定 全长NOSS和不同同工酶域的同工酶。这样的并行 结构-功能研究将确定共同特征和变异 在这三类诺斯中。对于每个一氧化氮合酶结构域或同工酶， 建议的工作对表达、纯化和生化 斯图尔实验室中晶体结构的表征 在Getzoff和Tainer实验室进行测定和分析。这个 从分子生物学，光谱，生物化学， 而结晶学实验将指导定点定向的设计 突变体及其辅因子、底物、中间体的选择 以及缓蚀剂复合体，以供进一步研究。这是集成的、递归的 该方法旨在增加对一氧化氮合酶催化的理解，并最终 帮助设计同工酶选择性一氧化氮合酶抑制剂，这将是无价的 用于在体内发现异构体功能的工具，并且是理想的 用于控制血压、感染性休克和 炎性损伤。

DESCRIPTION: The metalloenzyme nitric oxide synthase (NOS) regulates nitric oxide (NO) synthesis and thereby its biological activity. NO has a dual role as 1) a diffusible biological messenger for neurotransmission, long-term potentiation, platelet aggregation, and blood pressure regulation and 2) a cytotoxic agent for defense against tumor cells and intracellular parasites. NOS enzymes (NOSs), found in inducible (iNOS), constitutive endothelial (eNOS), and constitutive neuronal (nNOS) isoforms, achieve their important biological function by adopting an intriguing calcium-regulated catalytic mechanism and incorporating a unique assembly of five cofactors: heme, tetrahydrobiopterin (H4B), FMN, FAD and NADPH. NOSs generate NO by expending molecular oxygen and NADPH in each step of a two-step mechanism: first, a monooxygenase-like reaction converts L-arginine to the intermediate N4-hydroxy-L- arginine (NOH-arg), then an unprecedented reaction converts NOH-arg to citrulline and NO. To understand in atomic detail the unique structural metallobiochemistry of NOSs, integrated crystallographic and biochemical studies are proposed for the three major classes of NOS enzymes. Each NOS subunit is divided into two domains joined by a calmodulin-binding hinge region: an oxygenase domain with heme, H4B, and L-arginine binding sites forming the catalytic center for NO production, and a reductase domain with NADPH, FAD, and FMN binding sites supplying electrons to the heme. Electron transfer from the flavins to the heme is controlled by calmodulin (CaM), which fulfills a novel role for a calcium binding protein. The proposed studies first aim to characterize the structural biochemistry for individual oxygenase and reductase domains, which have been overexpressed and crystallized, and then to use these results to determine structures of full-length NOSs and of different analogous isozyme domains. Such parallel structure-function studies will establish common features and variations among these three classes of NOSs. For each NOS domain or isozyme, the proposed work couples expression, purification, and biochemical characterization in the Stuehr laboratory with crystallographic structure determination and analysis in the Getzoff and Tainer laboratories. The results from these coupled molecular biological, spectroscopic, biochemical, and crystallographic experiments will guide the design of site-directed mutants and the selection of appropriate cofactor, substrate, intermediate and inhibitor complexes for further research. This integrated, recursive approach aims to increase understanding of NOS catalysis and ultimately to aid the design of isozyme-selective NOS inhibitors, which will be invaluable tools for discovering isoform functions in vivo and are desirable as therapeutic agents for controlling blood pressure, septic shock, and inflammatory damage.