STRUCTURE-FUNCTION OF CYTOCHROME P-450

细胞色素 P-450 的结构-功能

• 批准号: 3838351
• 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/3838351
• 关键词: benzopyrenes; chemical binding; chemical cleavage; conformation; crosslink; cytochrome P450; cytochrome b; enzyme mechanism; enzyme structure; enzyme substrate; intermolecular interaction; laboratory rat; liver; membrane proteins; membrane structure; microsomes; protein purification; protein sequence; protein structure function; proteolysis; thermodynamics

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.

本项目的总体目标是阐明结构-功能 哺乳动物细胞色素P450的亲缘关系。这样的信息将 提高对P450在细胞代谢中的作用的理解 内源和环境化学品，并帮助发展 特定的P450抑制剂。研究内容主要集中在1)结构上 单独的P450，2)P450与其他膜的相互作用 蛋白质和3)P450-底物相互作用。1)膜的表面形貌 涉及P450形式1A1、2B1和2E1的研究揭示了一种常见的蛋白质组分- 与预测开启P450相对应的高度敏感区域 浮出水面。这一发现与公共第三级的概念是一致的 不同类别哺乳动物P450的结构。底物结合 用切割的P450进行的实验表明，P450的结构完整性 该区域是P450 2B1与苯丙胺结合的关键区域，但不是 对于苯并[a]芘与P450 1A1的结合。我们正在利用实验数据 结合计算机辅助多序列比对和 建立哺乳动物三级结构模型的分子模拟 P450。2)我们以前对P450的四元结构进行了研究 延伸以检查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 表格2B1及2E1，但不包括1A1。P450基因的多序列比对 建议了几种碱性氨基酸，它们可能对结合 细胞色素b5。P450-P450相互作用的研究 苯并[a]芘作为活性中心结构探针与P450 1A1结合。 而抗该P450的单抗1-7-1对结合没有影响， 抗体介导的P450s的交联减少了结合。这个结果 提示P450活性可能受四元组相互作用的调节 是P450的。3)用抗P450 2B1的单抗确定 苯非他明与大鼠肝微粒体中该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显示了膜相 在20摄氏度时的转变数据显示了薄膜的影响 P450与底物相互作用的结构。