STRUCTURE-FUNCTION OF CYTOCHROME P-450

本课题的总体目标是阐明结构-功能 哺乳动物细胞色素P450的关系。这些信息将 提高我们对P450在代谢中的作用的理解， 内源性和环境化学物质，并帮助发展 特异性P450抑制剂。研究的重点是1）结构 单个P450，2）P450与其他膜之间的相互作用 蛋白质和3）P450-底物相互作用。1)一种膜形貌 一项涉及P450 1A 1、2B 1和2 E1型的研究显示， 与P450上的预测转向相对应的敏感区域 面这一发现与共同的三级结构的概念是一致的。 不同种类的哺乳动物P450的结构。底物结合 用裂解的P450进行的实验表明， 该区域对于P450 2B 1与苄非他明的结合是关键的，但不是 苯并[a]芘与P450 1A 1的结合。我们利用实验数据 结合计算机辅助多序列比对， 分子建模，以开发哺乳动物的三级结构模型 P450。2)我们以前对P450四级结构的研究， 扩展到检查P450-细胞色素b5相互作用的特异性。 P450的免疫纯化显示细胞色素b5与P450结合 形式2B 1和2 E1，但不是1A 1。P450的多序列比对 提出了几种碱性氨基酸，这些氨基酸可能在结合 细胞色素b5。P450-P450相互作用通过使用 苯并[a]芘结合P450 1A 1作为活性位点结构探针。 虽然针对该P450的单克隆抗体1-7-1对结合没有影响， 抗体介导的P450交联降低了结合。这一结果 表明P450活性可能受四级相互作用的调节 P450的3)用抗P450 2B 1单克隆抗体鉴定P450 2B 1的表达。 在大鼠肝微粒体中，苄非他明与该P450的相互作用， 多个P450的存在该反应的热力学参数 和膜流动性的测量都揭示了膜相 在20摄氏度下转变。数据显示膜的影响 P450-底物相互作用。

The overall goal of this project is the elucidation of structure-function relationships for the mammalian cytochrome P450s. Such information will improve our understanding of the role of P450s in the metabolism of endogenous and environmental chemicals, and aid the development of specific P450 inhibitors. The research is focused on 1) structure of individual P450s, 2) interactions between P450s and other membrane proteins and 3) P450-substrate interactions. 1) A membrane topography study involving P450 forms 1A1, 2B1 and 2E1 revealed a common proteoly- tically sensitive region which corresponds to a predicted turn on the P450 surface. This finding is consistent with the concept of a common tertiary structure for different classes of mammalian P450s. Substrate binding experiments with the cleaved P450s showed that the structural integrity of this region is critical for binding of P450 2B1 to benzphetamine, but not for benzo[a]pyrene binding to P450 1A1. We are utilizing experimental data in conjunction with computer-assisted multiple sequence alignment and molecular modeling to develop a tertiary structure model of mammalian P450. 2) Our previous studies on quaternary structure of P450s have been extended to examine the specificity of the P450-cytochrome b5 interaction. Immuno-purification of P450 revealed binding of cytochrome b5 to P450 forms 2B1 and 2E1, but not 1A1. Multiple sequence alignment of P450s suggested several basic amino acids which may be important in binding to cytochrome b5. P450-P450 interactions were examined by using benzo[a]pyrene binding to P450 1A1 as an active site structural probe. While monoclonal antibody 1-7-1 to this P450 had no effect on binding, antibody-mediated cross-linking of P450s reduced binding. This result indicates that P450 activities may be modulated by quaternary interactions of P450s. 3) A monoclonal antibody to P450 2B1 was used to define the interaction of benzphetamine with this P450 in rat liver microsomes, in the presence of multiple P450s. Thermodynamic parameters for this reaction and membrane fluidity measurements both revealed a membrane phase transition at 20 degrees C. The data shows the influence of membrane structure on P450-substrate interactions.