STRUCTURE AND CHARACTERIZATION OF CYTOCHROME P-450

细胞色素 P-450 的结构和表征

• 批准号: 3874645
• 负责人: F K FRIEDMAN
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3874645
• 关键词: NADPH cytochrome c2 reductase; benzopyrenes; carbopolycyclic compound; chemical binding; chemical carcinogen; chemical carcinogenesis; chemical structure function; crosslink; cytochrome P450; electron spin resonance spectroscopy; enzyme complex; enzyme induction /repression; enzyme linked immunosorbent assay; enzyme mechanism; enzyme structure; enzyme substrate; flash photolysis; fluorescence spectrometry; isozymes; laboratory mouse; laboratory rat; liver metabolism; membrane proteins; microsomes; molecular site; monoclonal antibody; oxidoreductase; protein structure function; steroid hormone metabolism; testosterone; toxin metabolism; unspecific monooxygenase

The focus of this project is the characterization of structure-function relationships the cytochromes P-450. A cross-linking study of intact rat liver microsomal membranes revealed that P-450c, which metabolizes polycyclic hydrocarbons, is specifically associated with both P-450 reductase as well as P450 2a, which metabolizes testosterone. These results support the membrane cluster model in which P-450s and P-450 reductase both exist as stable complexes rather that as monomers. Such specific P-450 interactions may influence the secondary metabolism of P-450 substrates. The active site structure of P-450c was examined in binding studies using the substrate benzopyrene (BP). BP fluorescence was quenched upon binding to either microsomal or purified P-450c. The fluorescence of the BP-P-450c complex was used to probe the interaction between BP substrate and active site heme. Addition of P-450 reductase promoted a closer association of heme and BP, which demonstrates that the reductase does not function solely as an electron carrier. Flash photolysis experiments of CO recombination with the P-450 heme yielded parallel kinetic data on the effect of BP on active site dynamics. A three dimensional model of mammalian P-450 is being developed using both theoretical and experimental approaches. The latter includes identification of exposed surface regions on P-450s by protease digestion experiments, and the use of antibodies to synthetic P-450 peptides to identify functionally significant sequences.

本项目的重点是结构-功能的表征 细胞色素P-450的相互关系。正常大鼠的交联性研究 肝微粒体膜显示，代谢的P-450c 多环烃，与P-450特别相关 还原酶以及代谢睾酮的P450 2a。这些结果 支持P-450s和P-450都有还原酶的膜团模型 以稳定的络合物而不是单体的形式存在。这种特殊的P-450 相互作用可能影响P-450底物的次生代谢。 在结合研究中对P-450c的活性中心结构进行了研究 底物苯并苯(BP)。BP荧光在结合时被猝灭 微粒体或纯化的P-450c。BP-P-450C的荧光性质 用络合物研究了BP底物与活性物质的相互作用 站点血红素。P-450还原酶的加入促进了更紧密的结合 血红素和BP，这表明还原酶不是单独起作用的 作为电子载体。CO复合的闪光光解实验 与P-450的血红素产生平行的动力学数据，关于BP对 活动站点动态。哺乳动物P-450的三维模型正在进行中 用理论和实验两种方法开发。后者 包括用酶对P-450表面暴露区域的鉴定 消化实验，以及合成P-450抗体的使用 识别具有重要功能的序列的多肽。