MOLECULAR BASIS OF HUMAN CYTOCHROME P450 3A FUNCTION

描述：本提案的长期目标是阐明 细胞色素P450独特功能特性的结构基础 3A. 这些酶是非常通用的催化剂，并在以下方面发挥关键作用： 多种药理学和药理学化合物的肝代谢 毒理学兴趣。 P450 3A酶被诱导或抑制， 许多外来化合物，并涉及重要的药物-药物 人类的互动。 细胞色素P450 3A的功能 在物种内和物种间是保守的，但与P450不同 从其他的亚家族。 大多数P450 3A酶催化类固醇6 β-羟基化和大环内酯类抗生素代谢， α-萘酮（α-NF）刺激。 细胞色素P450 3A 可容纳任何P450已知的一些最大基板，例如 环孢菌素A，并且被认为具有多个结合位点。 然而，在这方面， 关于酶的结构特征的信息很少 赋予其催化性能。 建立在广泛的现场导向 诱变和分子建模研究，从这个实验室对 P450 2B特异性的决定因素，目前的建议集中在人类 P450 3A 4和3A 5。 3A 4是P450在肝脏中最高表达的基因。 大多数人，似乎代谢更多的临床重要药物， 任何其他人类P450 P450 3A 4还代谢环境中的 污染物苯并（a）芘和黄曲霉毒素B1。 P450 3A 5表达于 大约四分之一的人的肝脏。 核心假设是 细胞色素P450 3A具有结构上不同的底物结合， 效应位点。 具体目的是：1）探讨 在P450家族2酶中鉴定出的底物识别位点在调节 底物特异性和人P450 3A 4的α-NF刺激， 2）使用随机诱变结合功能筛选， 鉴定负责P450 3A 4的α-NF刺激的残基，和 探索关键蛋白类黄酮反应性改变的生化基础 盒、定点和随机突变体; 3）定位底物 P450 3A 4和3A 5的3-D同源性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述应 有助于预测药物-药物相互作用并导致改进的药物治疗。

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.