PARTICIPATION OF CYTOCHROME B5 IN ANESTHETIC METABOLISM

细胞色素5可增加，减少或无影响底物代谢， 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催化的氧化作用困扰了研究人员几十年。与 内源性化合物和药物的数量最近显著增加 在细胞色素存在的情况下， B5，这个问题变得越来越有趣，也越来越具有生物学意义。 这项提案的长期目标是了解分子 基于细胞色素P450催化代谢的显著刺激， 某些底物，并确定生理 这个反应的意义。这个问题将通过以下方式解决： 阐明了模型化合物甲氧氟烷（MF）， 一种挥发性麻醉剂，诱导对细胞色素B5的需求， 细胞色素P450 2B4（LM2）代谢。这些研究可能最终导致 描述了术后并发症的病因和病理生理学 肝毒性归因于挥发性麻醉剂，并应 有助于我们理解细胞色素P450的氧化机制。 该提案的短期目标有三个方面。第一特定 目的：采用停流分光光度法，研究 底物MF和苄非他明对CytP450还原酶结合的影响 和细胞色素b5对氧化亚铁细胞色素P450的还原率， oxyferrous cyt P450。产品的生产率 从底物结合位点释放的两种底物的量也将 追究这些研究的解释将大大 通过计算光谱、电子结构和自旋 细胞色素P450中稳定和瞬时中间体的分布 反应周期，将与Gilda博士合作进行 勒夫 第二个具体目标是继续我们的突变研究， 在细胞色素P450氧化MF过程中，细胞色素b5的特定氨基酸。的 主要的兴趣将是两组残基，周围的酸性残基 参与结合其可溶性氧化还原的细胞色素B5的血红素 配偶体，和推定的蛋白质间结合的氨基酸 面 第三个具体目标是定位蛋白间结合 细胞色素P450上的细胞色素b5位点。 这将通过使用 细胞色素P450 2B4的模型，以选择表面上的氨基酸 细胞色素P450 2B4的表达可能参与了与细胞色素b5的相互作用。 然后将选择的氨基酸系统地突变为 使用丙氨酸扫描技术测定丙氨酸。 的能力 突变的细胞色素P450分子结合细胞色素b5并发挥功能 在电子转移与这些蛋白质将被评估。

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.