PARTICIPATION OF CYTOCHROME B5 IN ANESTHETIC METABOLISM

细胞色素 B5 参与麻醉代谢

• 批准号: 2177948
• 负责人: LUCY A WASKELL
• 金额: $ 20.44万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2177948
• 关键词: active sites; alanine; anesthetics; cytochrome P450; cytochrome b; cytochrome b5 reductase; drug metabolism; electron transport; enzyme activity; enzyme mechanism; enzyme substrate; laboratory rabbit; methoxyflurane; site directed mutagenesis; spectrometry; stoichiometry; stop flow technique; unspecific monooxygenase

Cyt 5 can increase, decrease or have no effect on substrate metabolism by cyt P450. How and why cyt b5 can have such an unpredictable effect on cyt P450 catalyzed oxidations has puzzled researchers for decades. With the recent marked increase in the number of endogenous compounds and drugs that have been shown to increase their metabolism in the presence of cyt b5, this question is becoming more interesting and biologically relevant. The long term objective of this proposal is to understand the molecular basis of the marked stimulation of the cyt P450 catalyzed metabolism of certain substrates by cyt b5 and to determine the physiological significance of this reaction. The problem will be addressed by elucidating the mechanism by which the model compound methoxyflurane (MF), a volatile anesthetic, induces the requirement for cyt b5 for its metabolism by cyt P450 2B4 (LM2). These studies may eventually lead to the delineation of the etiology and pathophysiology of the postoperative hepatotoxicity attributed to the volatile anesthetics, and should contribute to our understanding of the mechanism of oxidation by cyt P450. The short term goals of this proposal are three-fold. The first specific aim, using stopped-flow spectrophotometry, is to investigate the effect of the substrates, MF and benzphetamine on the binding of cyt P450 reductase and cyt b5 to oxyferrous cyt P450 and on the rate of reduction of oxyferrous cyt P450 by these two proteins. The rate at which the product of both substrates is released from the substrate binding site will also be investigated. The interpretation of these studies will be greatly facilitated by calculations of the spectra, electronic structure and spin distribution of stable and transient intermediates in the cyt P450 reaction cycle, which will be performed in collaboration with Dr. Gilda Loew. The second specific aim is to continue our mutation studies into the role of specific amino acids of cyt b5 in the oxidation of MF by cyt P450. Of major interest will be two groups of residues, the acidic residues around the heme of cyt b5 which are involved in binding to its soluble redox partners, and the amino acids on the putative inter-protein binding surface. The third specific aim will be to locate the interprotein binding site on cyt P450 for cyt b5. This will be accomplished using a model of cyt P450 2B4 to select which amino acids on the surface of cyt P450 2B4 might be involved in interactions with cyt b5. The selected amino acids will then be systematically mutated to alanine using the technique of alanine scanning. The ability of the mutant cyt P450 molecules to bind cyt b5 and to function in electron transfer with these proteins will be evaluated.

Cyt 5 可以通过以下方式增加、减少或不影响底物代谢： 细胞色素P450。 cyt b5 如何以及为何对 cyt 产生如此不可预测的影响 几十年来，P450 催化的氧化一直困扰着研究人员。随着 最近内源性化合物和药物的数量显着增加 已被证明在细胞色素存在的情况下可以增加新陈代谢 b5，这个问题变得越来越有趣并且与生物学相关。 该提案的长期目标是了解分子 显着刺激 cyt P450 催化代谢的基础 通过 cyt b5 检测某些底物并确定生理学 该反应的意义。该问题将通过以下方式解决 阐明模型化合物甲氧氟烷 (MF) 的作用机制， 一种挥发性麻醉剂，诱导其需要 cyt b5 通过 cyt P450 2B4 (LM2) 进行代谢。这些研究最终可能会导致 描述术后的病因和病理生理学 挥发性麻醉药的肝毒性，应 有助于我们了解 cyt P450 的氧化机制。 该提案的短期目标有三个。第一个具体 目的是使用停流分光光度法研究 底物、MF 和苯异丙胺对 cyt P450 还原酶结合的影响 和 cyt b5 至含氧铁细胞色素 P450 的减少率 氧化亚铁细胞色素P450由这两种蛋白产生。产品的速率 两种底物的从底物结合位点释放也将 被调查。这些研究的解释将极大地 通过光谱、电子结构和自旋的计算来促进 细胞色素 P450 中稳定和瞬时中间体的分布 反应循环，将与吉尔达博士合作进行 勒夫。 第二个具体目标是继续我们的角色突变研究 细胞色素 P450 氧化 MF 时细胞色素 b5 的特定氨基酸的变化。的 主要兴趣将是两组残基，即周围的酸性残基 cyt b5 的血红素参与与其可溶性氧化还原的结合 伙伴，以及推定的蛋白质间结合上的氨基酸 表面。 第三个具体目标是定位蛋白质间结合 cyt b5 位于 cyt P450 上的位点。 这将通过使用 cyt P450 2B4 模型来选择表面上的氨基酸 cyt P450 2B4 可能参与与 cyt b5 的相互作用。 然后，选定的氨基酸将被系统地突变为 丙氨酸使用丙氨酸扫描技术。 的能力 突变的 cyt P450 分子结合 cyt b5 并发挥作用 将评估这些蛋白质的电子转移。