STRUCTURE-FUNCTION OF CYTOCHROME P450

细胞色素 P450 的结构-功能

• 批准号: 5201473
• 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/5201473
• 关键词: Baculoviridae; NADPH cytochrome c2 reductase; binding proteins; carbon monoxide; carbopolycyclic compound; chemical binding; chemical kinetics; conformation; cytochrome P450; cytochrome b5 reductase; enzyme activity; enzyme substrate complex; flash photolysis; human genetic material tag; ligands; microsomes; molecular site; protein structure function; synthetic peptide

Structure-function relationships for the mammalian cytochromes P450 are examined. Specifically, we are elucidating the interactions between P450 and microsomal electron carrier proteins, substrates and ligands. We assessed the interaction of P450 with NADPH cytochrome P450 reductase to identify the specific amino acid residues involved in this protein- protein interaction. Molecular modeling in conjunction with P450 sequence alignments were used to predict the P450 binding site for reductase. Synthetic peptides corresponding to these regions on P450 2B1 were prepared and assessed for their ability to inhibit reductase-mediated P450 activities. A peptide corresponding to the C helix (residues 116- 134) inhibited benzphetamine demethylase, but had no effect when Arg-125 was replaced by Glu. In addition surface simulatory peptides were prepared which consist of sequences which are proximate in the tertiary structure but distant in primary sequence. A peptide consisting of elements of helices C and L was particularly effective in inhibiting several P450-mediated activities in both reconstituted and microsomal systems. These results thus identify both specific regions and a residue involved in P450 binding to reductase. The conformation and dynamics of P450 were examined with the CO flash photolysis technique. We evaluated the effect of a series of polycyclic aromatic hydrocarbons of varying sizes and shapes on the CO binding kinetics of both rat liver microsomal P450 1A1 and baculovirus expressed human P450 1A1. The results showed that substrates modulate CO binding by a dual mechanism involving both conformational and steric effects. Experiments with baculovirus expressed human P450 3A4, a form which metabolizes many important drugs with diverse structures, showed that this P450 consists of a population of conformers that differ in their substrate recognition properties. This finding helps explain the broad substrate specificity of this P450. These results demonstrate the utility of CO binding kinetics as a probe of P450 structure/dynamics in a native membrane environment.

哺乳动物细胞色素P450的结构与功能关系如下 检查过了。具体来说，我们正在阐明P450之间的相互作用 和微粒体电子载体蛋白、底物和配体。我们 评估P450与NADPH细胞色素P450还原酶的相互作用 确定这种蛋白质中涉及的特定氨基酸残基- 蛋白质相互作用。结合P450序列的分子模拟 序列比对用于预测还原酶的P450结合位点。 与P450 2B1上这些区域相对应的合成肽是 制备并评估其抑制还原酶介导的能力 P450活性。与C螺旋相对应的多肽(残基116- 134)抑制苯丙胺去甲基酶，但不抑制Arg-125的作用 被Glu取代。此外，表面模拟多肽还 由第三系中最接近的序列组成的 结构，但在初级序列上相距较远。一种由以下成分组成的肽 螺旋C和L的成分对抑制作用尤为有效 重组细胞和微粒体中几种P450介导的活性 系统。因此，这些结果既能识别特定区域，又能识别残基 参与P450与还原酶的结合。分子的构象和动力学 用CO闪光光解技术检测P450。我们评估了 一系列不同结构的多环芳烃的作用 大小和形状对两种大鼠肝微粒体一氧化碳结合动力学的影响 P450-1A1和杆状病毒表达人P450-1A1。结果显示， 底物通过双重机制调节CO结合，包括 构象效应和空间效应。用杆状病毒表达的实验 人类P450 3A4，一种能代谢许多重要药物的形式 不同的结构，表明这个P450由一个种群组成 底物识别特性不同的异构体。这 这一发现有助于解释该P450的广泛底物特异性。这些 结果表明，CO结合动力学可作为P450的探针 原生膜环境中的结构/动力学。