PARTICIPATION OF CYTOCHROME B5 IN ANESTHETIC METABOLISM

细胞色素P450（cyts P450）是一个普遍存在的混合功能氧化酶超家族，也被称为大自然的喷灯。 在哺乳动物中，细胞色素P45 O可能占肝微粒体蛋白的10%，并且已知其代谢a。在纯化的重组系统中，称为细胞色素b5（cyt b5）的第二种微粒体蛋白可以增加、减少或不影响cyt P450的底物代谢。细胞色素b5如何以及为什么能对细胞色素P45 O催化的氧化产生如此不可预测的影响，几十年来一直困扰着研究人员。随着近年来内源性化合物和药物的数量显著增加，这些化合物和药物在细胞色素b5存在的情况下被证明可以增加其代谢，这个问题变得更加有趣和生物相关。本提案的长期目标是了解细胞色素B5对某些底物的细胞色素P450催化代谢的显著刺激的分子基础，并确定该反应的生理意义。该问题将通过阐明细胞色素b5增加细胞色素P450 2B 4（LM 2）催化的模型化合物甲氧氟烷（MF）（挥发性麻醉剂）和苄非他明的羟基化效率的机制来解决。这些研究可能最终提供洞察的病因学和病理生理学的术后肝毒性归因于挥发性麻醉剂，如氟烷，和设计更安全的麻醉剂。该提案的短期目标有三个方面。第一个具体的目的是调查是否细胞色素b5或细胞色素P450还原酶减少氧化亚铁细胞色素P450 2B 4更迅速。细胞色素B5比细胞色素P45 O还原酶更快地还原氧化亚铁细胞色素P450，这可以解释细胞色素B5介导的超氧化物生成减少和细胞色素P45 O周转期间产物形成增加。为了进一步探讨细胞色素b5增加细胞色素P45 O的催化效率的机制，我们最近通过鉴定突变的细胞色素P45 O和细胞色素b5，它们缺乏与各自的氧化还原配偶体结合的能力，来描绘细胞色素P450-细胞色素b5蛋白间结合位点。第二个具体目标将是表征这些突变蛋白质结合其氧化还原伙伴的能力，并在不同的氧化态进行电子转移反应。为了确定在蛋白间复合物中哪个cyt b5和cyt P45 O残基接触，还将检查成对突变体的结合能力。第三个具体目标将是描绘的蛋白间结合位点之间的膜结合结构域的细胞色素b5和细胞色素P45 O 2B 4使用定点诱变系统突变的氨基酸在这两种蛋白质的膜锚。 实验提出，这也将评估这种膜内结合相互作用的特异性。

The cytochromes P450 (cyts P450) are an ubiquitous superfamilv of mixed function oxidases which are also known as Mother Nature's blowtorch. In mammals, cyts P45O may account for up to 10% of the hepatic microsomal proteins and are known to metabolize a. majority of the xenobiotics including drugs and carcinogens encountered by man. In purified reconstituted systems, a second microsomal protein known as cytochrome b5 (cyt b5) 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 P45O 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 cyt b5 increases the efficiency of the cyt P450 2B4 (LM2) catalyzed hydroxylation of the model compounds methoxyflurane (MF), a volatile anesthetic, and benzphetamine. These studies may eventually provide insight into the etiology and pathophysiology of the postoperative hepatotoxicity attributed to the volatile anesthetics, such as halothane, and the design of safer anesthetics. The short-term goals of this proposal are three-fold. The first specific aim is to investigate whether cyt b5 or cyt P450 reductase reduces oxyferrous cyt P450 2B4 more rapidly. More rapid reduction of oxyferrous cyt P450 by cyt b5 than by cyt P45O reductase would explain the cyt b5-mediated decrease in the production of superoxide and increase in product formation during cyt P45O turnover. To further probe the mechanism by which cyt b5 increases the catalytic efficiency of cyt P45O, we have recently delineated the cyt P450-cyt b5 interprotein binding site by identifying mutant cyts P45O and cyts b5 which are deficient in their ability to bind to their respective redox partners. The second specific aim will be to characterize these mutant proteins with respect to their ability to bind their redox partners and undergo electron transfer reactions in different oxidation states. In an effort to determine which cyt b5 and cyt P45O residue are in contact in the interprotein complex, the ability of pairs of mutants to bind will also be examined. The third specific aim will be to delineate the interprotein binding site between the membrane binding domains of cyt b5 and cyt P45O 2B4 using site-directed mutagenesis to systematically mutate amino acids in the membrane anchors of these two proteins. Experiments are proposed which will also evaluate the specificity of this intramembrane binding interaction.