SELECTIVE DESTRUCTION OF CYTOCHROME P450 BY DRUGS

细胞色素P450和一氧化氮合酶是硫酸盐连接的血红蛋白。细胞色素P450在亲脂性外源物质的代谢和内源性脂质因子的生物合成中起着关键作用，其中包括20-羟基二十碳四烯酸(20-HETE)。20-HETE参与了血管压力的调节。一氧化氮合酶产生一氧化氮，这是一种气体分子，具有多种功能，如神经递质、血管调节因子和抗感染剂。这两个酶系统通过它们的机制、它们催化的化学作用以及NO和20-HETE协调的血管调节作用而联系在一起。我们建议继续我们对细胞色素P450和一氧化氮合酶的结构、机制和功能的分析。以细胞色素P450为例，我们计划通过绘制活性部位残基，制备哺乳动物和细菌P450酶的嵌合体，阐明嗜热P450(CYP119)的结构和功能，以及继续对底物专一性的决定因素进行实验和计算分析，来阐明P450酶活性部位的结构及其与底物专一性的关系。我们还将通过确定其链长的基础和omega-羟化区域特异性，通过开发可在体内用于研究脂肪酸omega-羟化的生理作用的CYP4A亚型特异性抑制剂，通过阐明所有已知的大鼠和人类CYP4A亚型的不同底物特异性，对P450酶的CYP4A家族进行详细的研究。在对一氧化氮合酶的所有平行研究中，我们建议利用嵌合体、定点突变、修复基团替换和复杂的光谱技术来研究三种一氧化氮合酶亚型的结构和机制，重点是电子传递途径和四氢生物蝶呤的作用。我们还将描述一氧化氮异构体中的二聚体界面，阐明负责钙调蛋白与三种异构体的不同结合的接触，并探索将二聚体接触区用作开发异构体特异性一氧化氮合酶抑制剂的可能性。

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.