SELECTIVE DESTRUCTION OF CYTOCHROME P450 BY DRUGS

细胞色素P450和一氧化氮合酶是巯基连接的血红素蛋白。细胞色素P450在亲脂性外源物质的代谢和内源性脂质因子（包括20-羟基二十碳四烯酸（20-HETE））的生物合成中起关键作用。20-HETE参与血管压力的调节。一氧化氮合酶产生NO，一种具有许多功能的气体分子，如神经递质、血管调节因子和抗感染剂。这两种酶系统通过它们的机制、它们催化的化学反应以及NO和20-HETE的协调血管调节作用而联系在一起。我们建议继续我们的细胞色素P450和一氧化氮合酶的结构，机理和功能分析。在细胞色素P450的情况下，我们计划阐明的P450酶活性位点的结构和它们的关系，底物特异性映射活性位点残基，制备哺乳动物和细菌的P450酶的嵌合体，阐明嗜热P450（CYP 119）的结构和功能，并继续实验和计算分析的底物特异性的决定因素。我们还将通过确定其链长和ω-羟基化区域特异性的基础，通过开发可用于体内研究脂肪酸ω-羟基化生理作用的CYP 4A亚型特异性抑制剂，通过阐明所有已知大鼠和人CYP 4A亚型的差异底物特异性，对CYP 4A家族的P450酶进行详细研究。在所有的平行研究的一氧化氮合酶，我们建议调查的结构和机制的三个一氧化氮合酶亚型，重点是电子转移途径和四氢生物蝶呤的作用，使用嵌合体，定点诱变，假体组替换，和复杂的光谱技术。我们还将表征二聚体接口的一氧化氮亚型，澄清负责钙调素的差异结合的三种亚型的接触，并探讨使用二聚体接触区域作为目标的亚型特异性一氧化氮合酶抑制剂的发展的可能性。

The cytochromes P450 and nitric oxide synthases are thiolate-ligated hemoproteins. The cytochromes P450 play key roles in metabolism of lipophilic xenobiotics and the biosynthesis of endogenous lipid factors, including 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is involved in regulation of vascular pressure. The nitric oxide synthases produce NO, a gaseous molecule with many functions as a neurotransmitter, vascoregulatory factor, and anti-infective agent. The two enzyme systems are linked by their mechanisms, the chemistry they catalyze, and the coordinate vascoregulatory actions of NO and 20-HETE. We propose to continue our structural, mechanistic, and functional analysis of both the cytochrome P450 and nitric oxide synthases. In the case of cytochrome P450, we plan to elucidate the structures of P450 enzyme active sites and their relationship to substrate specificity by mapping active site residues, preparing chimeras of mammalian and bacterial P450 enzymes, elucidating the structure and function of a thermophilic P450 (CYP119), and continuing an experimental and computational analysis of the determinants of substrate specificity. We will also carry out a detailed investigation of the CYP4A family of P450 enzymes by determining the basis for their chain length and the omega- hydroxylation regiospecificities, by developing CYP4A isoform-specific inhibitors that can be used in vivo to study the physiological roles of fatty acid omega-hydroxylation, by elucidating the differential substrate specificities of all the known rat and human CYP4A isoforms. In all parallel studies of the nitric oxide synthases we propose to investigate the structure and mechanism of the three nitric oxide synthase isoforms, with emphasis on the electron transfer pathway and the role of tetrahydrobiopterin, using chimeras, site specific mutagenesis, prosthetic group replacement, and sophisticated spectroscopic techniques. We will also characterize the dimer interfaces in the nitric oxide isoforms, clarify the contacts responsible for the differential binding of calmodulin to the three isoforms, and explore the possibility of using the dimer contact regions as a target for the development of isoform-specific nitric oxide synthase inhibitors.