HEPATIC MICROSOMAL DRUG OXIDATION

肝微粒体药物氧化

• 批准号: 2171043
• 负责人: RONALD W ESTABROOK
• 金额: $ 26.77万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-09-01 至 1997-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2171043
• 关键词: Escherichia coli; NADPH cytochrome c2 reductase; chimeric proteins; computer simulation; crosslink; cytochrome P450; drug metabolism; electron transport; enzyme inhibitors; gene expression; high performance liquid chromatography; liver metabolism; microsomes; molecular site; oxidation; polymerase chain reaction; protein engineering; protein structure function; recombinant proteins; spectrometry; steroid hormone metabolism; toxin metabolism

The hemoproteins called cytochrome P450 function in the oxygenation of steroids, fatty acids, and fat soluble vitamins, to name but a few examples. Most important is the role of P45Os in the metabolism of many xenobiotics where they may generate reactive metabolites that have toxic effects, in particular drugs, pollutants, food additives etc. The number of P45Os cloned and sequenced now exceeds 200; the majority of these are present in mammalian tissues. In recent years the heterologous expression of specific P45Os has facilitated their enzymatic characterization permitting the study of structure/function relationships. Recently, this laboratory has successfully engineered two artificial fusion proteins containing the heme domain of specific P45Os linked to the flavoprotein domain of NADPH-P450 reductase and expressed these in high yield in E. coli. Methods have been developed for the purification to homogeneity of these enzymatically active fusion proteins. It is proposed to study the mechanism of domain interactions by engineering fusion proteins containing different reductase moieties (rat, human, plant, bacterial (BM3) and yeast) with different P45Os (human lA2, 2D6, 3A4 and 4A9). fusion proteins will be expressed containing linkers of different length, amino acid charge, and hydrophobicity. The goal is to identify the role of specific amino acids, presumably on the surface of the flavoprotein reductase and/or the specific P45Os, that limit the rate of electron transfer from the flavin domain to the heme domain. Specific amino acids of the P450 or reductase will be modified by mutagenesis to confirm their critical roles in domain interaction with the objective of developing methods for enhancing electron transfer. Experiments will be carried out designed to engineer fusion proteins containing three or more functional proteins required for P450 activity, such as the preparation of a three component fusion protein containing P45017A, NADPH-P450 reductase and cytochrome b5. The effect of structure modifications, will be measured by determining patterns of substrate metabolism, the tight coupling of NADPH oxidation with substrate metabolism, the spectrophotometric measurement of steady state reduction of the flavin and heme components of a fusion protein, and the stopped-flow analysis of P450 reduction. Availability of large amounts of purified P450 proteins also offers the opportunity to initiate attempts to crystallize a mammalian P450.

称为细胞色素 P450 的血红素蛋白在氧合中发挥作用 类固醇、脂肪酸和脂溶性维生素，仅举几例 例子。最重要的是 P45Os 在许多代谢中的作用 异生物质可能产生有毒的反应性代谢物 影响，特别是药物、污染物、食品添加剂等。数量 目前已克隆并测序的 P45O 数量已超过 200 个；其中大多数是 存在于哺乳动物组织中。近年来异源表达 特定 P45O 的存在促进了它们的酶学表征 允许研究结构/功能关系。最近，这个 实验室成功设计出两种人工融合蛋白 含有与黄素蛋白连接的特定 P45O 的血红素结构域 NADPH-P450 还原酶结构域，并在大肠杆菌中高产表达。 大肠杆菌。已开发出纯化均质的方法 这些具有酶活性的融合蛋白。建议研究 通过工程融合蛋白包含结构域相互作用的机制 不同的还原酶部分（大鼠、人类、植物、细菌 (BM3) 和 酵母）与不同的 P45O（人 IA2、2D6、3A4 和 4A9）。融合蛋白 将表达含有不同长度、氨基酸的接头 电荷和疏水性。目标是确定具体的角色 氨基酸，可能位于黄素蛋白还原酶的表面 和/或特定的 P45O，限制电子转移速率 黄素结构域至血红素结构域。 P450 的特定氨基酸或 还原酶将通过诱变进行修饰以确认其关键作用 在领域互动中，以开发方法为目标 增强电子转移。将进行实验旨在 工程融合蛋白含有三种或更多功能蛋白 P450活性所需的，例如三组分的制备 含有P45017A、NADPH-P450还原酶和细胞色素b5的融合蛋白。 结构修改的效果将通过确定来测量 底物代谢模式、NADPH 氧化的紧密耦合 随着底物代谢，稳定的分光光度测量 融合蛋白的黄素和血红素成分的状态减少，以及 P450还原的停流分析。大量供货 纯化的 P450 蛋白也提供了启动尝试的机会 使哺乳动物 P450 结晶。