STRUCTURAL STUDIES ON NITRIC OXIDE SYNTHASE

本项目的长期目标是研究结构-功能 一氧化氮合酶（NOS）。 NOS是酶 负责精氨酸氧化成一氧化氮（NO）。 在 近年来，一氧化氮被认为是一种主要的生理活性物质， 参与神经、免疫和心血管系统的信使分子 系统. 由于NO作为一种调节剂的效力和重要性， 分子，NOS是一种严格控制下的复杂酶。 酶 由一个血红素结构域组成，精氨酸的实际氧化发生在该结构域中 和FMN/FAD结构域，其用于将电子从NADPH穿梭至 血红素结构域 在血红素和黄素结构域之间是一个连接体， 另一种调节分子钙调素。 除了血红素，FMN，和 FAD，NOS含有另一种功能未知的辅因子， 四氢生物蝶呤。 序列比对清楚地表明， 结构域与细胞色素P450还原酶非常相似，但血红素 结构域与其他血红素蛋白几乎没有相似之处， 与P450在功能和光谱性质上相似。 我们的目标 确定NOS的三种主要形式的晶体结构： 巨噬细胞诱导型或iNOS、神经元型或nNOS和内皮型或eNOS。 由于最独特的特征是血红素域， 域是合理药物设计的主要目标，我们最初有 专注于结晶这三种物质的血红素域 的晶体 eNOS血红素结构域与nNOS的2.5A和更小晶体结合， 已经获得了iNOS血红素结构域。 我们最初的目标是解决 eNOS血红素结构域的晶体结构。

The long range goal of this project is to study structure-function relationships in nitric oxide synthase (NOS). NOS is the enzyme responsible for the oxidation of arginine to nitric oxide (NO). In recent years nitric oxide has been recognized as a major physiological messenger molecule involved in the nervous, immune, and cardiovascular systems. Owing to the potency and importance of NO as a regulatory molecule, NOS is a complex enzyme under stringent control. The enzyme consists of a heme domain where the actual oxidation of arginine occurs and an FMN/FAD domain that serves to shuttle electrons from NADPH to the heme domain. Between the heme and flavin domains is a linker that binds another regulator molecule, calmodulin. In addition to heme, FMN, and FAD, NOS contains yet another cofactor of unknown function, tetrahydrobiopterin. Sequence alignments clearly show that the flavin domain is very similar to cytochrome P450 reductase but that the heme domain bears little resemblance to other heme proteins despite the similarity to P450 in both function and spectral properties. Our goal is to determine the crystal structure the three primary forms of NOS: macrophage inducible or iNOS, neuronal or nNOS and endothelial or eNOS. Since the most unique feature is the heme domain and since the heme domain is the primary target for rational drug design, we initially have focused on crystallizing the heme domain of all three. Crystals of the eNOS heme domain diffract to 2.5A and smaller crystals of the nNOS and iNOS heme domains have been obtained. Our initial goal will be to solve the crystal structure of the eNOS heme domain.