MOLECULAR BASIS OF HUMAN CYTOCHROME P450 3A FUNCTION

描述：这项建议的长期目标是澄清 细胞色素P450独特功能性质的结构基础 3A。这些酶是用途非常广泛的催化剂，在 多种药理活性物质的肝脏代谢研究 毒物学兴趣。P450 3A酶的诱导或抑制作用 大量外来化合物，涉及重要药物--药物 人类之间的相互作用。总的来说，细胞色素P450 3A的功能 在物种内和物种间是保守的，但与P450的不同 来自其他亚家族。大多数P450 3A酶催化类固醇6 β-羟基化与大环内酯类抗生素代谢及展示 α-萘黄酮(α-NF)刺激。细胞色素P450 3A 适应一些已知的任何P450的最大底物，例如 环孢素A，并被认为具有多个结合位点。然而， 关于酶的结构特征的信息很少 这赋予了它们催化性能。在广泛的现场导向的基础上建造 来自本实验室的诱变和分子模拟研究 P450 2B特异性的决定因素，目前的建议侧重于人类 P450 3A4和3A5。3A4是P450在肝脏中表达最高的基因 大多数人类和似乎代谢更多临床上重要的药物比 任何其他人类的P450。P450 3A4还代谢环境中的 污染物苯并(A)芘和黄曲霉毒素B1。P450 3A5表达于 大约每四个人中就有一个的肝脏。中心假设是 细胞色素P450 3A具有结构上不同的底物结合和 效应器位置。其具体目的是：1)探讨 调控中P450家族2酶底物识别位点的确定 人P450 3A4和人P450 3A4的底物特异性及α-核因子的刺激作用 3A5；2)将随机突变与功能筛选相结合 确定与P450 3A4的α-NF刺激有关的残基，以及 探讨KEY黄酮类反应性改变的生化基础 盒式、定点和随机突变体；3)底物定位 P450 3A4和3A5三维同源模型中的结合和效应位点。 人类P450 3A活性的结构决定因素的描绘应 帮助预测药物与药物之间的相互作用，并改进药物治疗。

DESCRIPTION: The long-term objective of this proposal is to elucidate the structural basis for the unique functional properties of cytochromes P450 3A. These enzymes are very versatile catalysts and play a crucial role in the hepatic metabolism of a wide variety of compounds of pharmacological and toxicological interest. P450 3A enzymes are induced or inhibited by numerous foreign compounds and are involved in important drug-drug interactions in humans. In general, the functions of cytochromes P450 3A are conserved within and across species but are distinct from those of P450s from other subfamilies. Most P450 3A enzymes catalyze steroid 6 beta-hydroxylation and macrolide antibiotic metabolism and exhibit stimulation by alpha-naphthoflavone (alpha-NF). Cytochromes P450 3A accommodate some of the largest substrates known for any P450, such as cyclosporin A, and are thought to possess multiple binding sites. However, little information is available, on the structural features of the enzymes that confer their catalytic properties. Building on extensive site-directed mutagenesis and molecular modeling studies from this laboratory on determinants of P450 2B specificity, the current proposal focuses on human P450 3A4 and 3A5. 3A4 is the most highly expressed P450 in the liver of most humans and appears to metabolize more clinically important drugs than any other human P450. P450 3A4 also metabolizes the environmental contaminants benzo(a)pyrene and aflatoxin B1. P450 3A5 is expressed in the liver of approximately one in four individuals. The central hypothesis is that cytochromes P450 3A have structurally distinct substrate binding and effector sites. The Specific Aims are to: 1) Probe the role of the substrate recognition sites identified in P450 family 2 enzymes in governing the substrate specificity and stimulation by alpha-NF of human P450 3A4 and 3A5; 2) Use random mutagenesis in conjunction with functional screening to identify residues responsible for alpha-NF stimulation of P450 3A4, and probe the biochemical basis of altered flavonoid responsiveness of key cassette, site-directed, and random mutants; 3) Localize the substrate binding and effector sites in 3-D homology models of P450 3A4 and 3A5. Delineation of the structural determinants of human P450 3A activity should aid in predicting drug-drug interactions and lead to improved drug therapy.