Investigation of Nitric Oxide Synthase Structure, Function and Inhibition

一氧化氮（NO）是多种生理和病理生理过程中重要的内源性信使。一氧化氮合酶（NOS）是由神经元（nNOS）、内皮（eNOS）和诱导（iNOS）三种不同基因编码的一氧化氮合酶（NOS）三种亚型合成的。一氧化氮在体内作为第二信使或细胞毒剂的双重作用表明了对不同一氧化氮同种异构体的严格调控的重要性。NOS的每个亚基由一个包含NADPH结合位点的还原酶结构域、两个黄素和一个包含血红素、l -精氨酸和四氢生物蝶呤结合位点的加氧酶结构域组成。这两个结构域由钙调素（CaM）结合结构域连接，该结构域在酶的激活中起重要作用。CaM是钙离子的主要蛋白质介质，也是一个重要的次级信使。CaM经历钙依赖的构象变化，使其能够结合并激活包括所有三种NOS同工酶在内的许多靶蛋白。本研究计划的两个长期目标是：1)了解CaM结合和控制所有三种NOS同工酶的机制；2)研究人类NOS同工酶的选择性配体结合及抑制作用。

Nitric oxide (NO) is an important endogenous messenger in a variety of physiological and pathophysiological processes. NO is synthesized by the three isoforms of nitric oxide synthase (NOS) that are encoded by different genes, neuronal (nNOS), endothelial (eNOS) and inducible (iNOS). The dual role of NO in the body as a second messenger or cytotoxic agent shows the importance for tight regulation of the different NOS isoforms. Each subunit of NOS is composed of a reductase domain that contains the binding sites for NADPH, and two flavins as well as an oxygenase domain that contains the binding sites for heme, L-arginine and tetrahydrobiopterin. The two domains are linked by a calmodulin (CaM) binding domain that plays an important role in the activation of the enzyme. CaM is the primary protein mediator of the calcium cation that is also an important secondary messenger. CaM undergoes calcium-dependent conformational changes that allow it to bind and activate scores of target proteins including all three NOS isozymes. Two of the long-term goals of this research program are to: 1) understand the mechanism(s) of CaM binding and control for all three NOS isozymes; 2) study the selective ligand binding and inhibition of human NOS isozymes. A critical structural determinant of NOS activity is the CaM controlled electron transfer from NADPH to the heme via two flavin cofactors. How CaM controls electron transfer is not fully understood and several differences have been noted between the three isoforms of NOS. Due to the large size and dynamic properties of the NOS enzymes, it is very difficult to get information on their full length protein structure. Structural information on NOS isozymes is limited to truncations encompassing their heme domains, reductase domains and the iNOS FMN subdomain bound to CaM. This is most likely due to the large conformational shift of the FMN subdomain believed to occur due to electron transfer during catalysis. As part of our proposed research program, we will perform NMR investigations on full-length as well as judiciously truncated forms of the enzymes to decipher the role of CaM and the different domains in the regulatory mechanism of the enzyme. These NMR experiments when combined with other biophysical studies and enzymes assays will provide a better understanding of the CaM dependent activation of NOS enzymes. The second major goal of our research program is to study selective binding and inhibition of human NOS isozymes. While considerable research has been performed on mammalian NOS enzymes, there is limited information on the human isozymes. We propose to use the human isozymes in our investigation and compare the results with previous studies performed on the corresponding mammalian enzymes. Our rational is based on the possibility that small differences between human and mammalian enzymes may be important in our search for isoform selective binding and enzyme inhibition.