STRUCTURE AND CHARACTERIZATION OF CYTOCHROME P-450

本项目的重点是结构-功能的表征 与细胞色素P-450的关系。完整大鼠的交联研究 肝微粒体膜显示，P-450 c，其代谢 多环烃，特别是与P-450 还原酶以及代谢睾酮的P450 2a。这些结果 支持膜簇模型，其中P-450和P-450还原酶都 以稳定的络合物而不是单体形式存在。这种特殊的P-450 相互作用可能影响P-450底物的次级代谢。 P-450 c的活性位点结构在结合研究中使用 底物苯并芘（BP）。BP荧光在结合后猝灭 微粒体或纯化的P-450 c。BP-P-450 c的荧光 复合物被用来探测BP底物和活性物质之间的相互作用 部位血红素。添加P-450还原酶促进了 血红素和BP，这表明还原酶并不单独起作用 作为电子载体。CO复合的闪光光解实验 与P-450血红素产生平行的动力学数据的影响，BP 活性位点动态哺乳动物P-450的三维模型正在 使用理论和实验方法开发。后者 包括通过蛋白酶鉴定P-450上暴露的表面区域， 消化实验，以及使用合成的P-450抗体 肽以鉴定功能上重要的序列。

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.