PARTICIPATION OF CYTOCHROME B5 IN ANESTHETIC METABOLISM

Cyt5可通过以下方式增加、减少或不影响底物代谢 细胞色素P450。Cyt b5如何以及为什么会对Cyt产生如此不可预测的影响 P450催化的氧化反应几十年来一直困扰着研究人员。与 最近内源性化合物和药物的数量明显增加 已被证明在Cyt存在下可以促进它们的新陈代谢 B5，这个问题变得越来越有趣，在生物学上也越来越相关。 这项提议的长期目标是理解分子 显著刺激细胞色素P450催化代谢的基础 用细胞色素b5测定某些底物的生理活性 这一反应的意义。这个问题将通过以下方式解决 阐明了模型化合物甲氧基氟烷(MF)， 一种易挥发的麻醉剂，导致对Cyt b5的需求 细胞色素P450 2B4(LM2)代谢。这些研究可能最终会导致 术后病因学和病理生理学的描述 肝毒性归因于挥发性麻醉剂，应该 有助于我们理解细胞色素P450的氧化机制。 这项提议的短期目标有三个方面。第一个具体 目的：采用停流分光光度法研究不同剂量黄连提取物对小鼠血清白蛋白含量的影响。 底物、甲硫氨酸和苯丙胺对细胞色素P450还原酶结合的影响 和细胞色素b5还原为含氧亚铁细胞色素P450 含氧细胞色素P450通过这两种蛋白表达。产品的生产速度 两种底物的结合部位也会从底物中释放出来 被调查。这些研究的解释将极大地 由光谱、电子结构和自旋的计算推动 稳定和瞬时中间产物在细胞色素P450中的分布 反应周期，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对该角色的突变研究 Cyt p450氧化MF过程中Cyt b5特定氨基酸的变化。的 主要关注的是两类残基，酸性残基周围 参与与其可溶性氧化还原结合的细胞色素b5的血红素 伴侣，以及推测的蛋白质间结合上的氨基酸 浮出水面。 第三个具体目标将是定位蛋白质间的结合 Cyt P450上的Cyt b5位点。这将通过使用 细胞色素P450 2B4模型选择表面哪些氨基酸 细胞色素P4502B4可能参与与细胞色素b5的相互作用。 然后，选定的氨基酸将被系统地突变为 丙氨酸采用丙氨酸扫描技术。的能力 突变的细胞色素P450分子与细胞色素b5结合并发挥作用 在与这些蛋白质的电子转移中，将进行评估。

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.