Participation of Cytochrome b5 in Anesthetic Metabolism

描述（由申请人提供）：细胞色素P450是一个普遍存在的混合功能氧化酶超家族，负责氧化大量化合物，包括人类使用的大多数药物。在我们研究细胞色素b5 （cyt b5）如何通过细胞色素P450 2B4 （cyt P450）刺激底物（如麻醉剂甲氧基氟醚）的代谢的过程中，我们最近首次测量了微粒体氧化亚铁细胞P450, 2B4通过细胞色素P450还原酶的还原速率。该实验发现，在cyt P450还原酶存在下，cyt P450 2B4的催化作用是通过还原的氧化亚铁中间体进行的。与此形成鲜明对比的是，在cyt b5存在的情况下，cyt P450 2134的催化作用发生迅速，没有形成可检测的中间体，从而减少了发生非生产副反应的时间。开发的氧化亚铁细胞P450 - 2B4还原速率测量工具将用于进一步探索氧化亚铁细胞P450的催化机制，了解氧化亚铁细胞P450 - 2B4如何促进药物代谢。本课题的短期目标是阐明cyt b5和cyt P450还原酶对cyt P450 2B4催化作用的显著不同作用的分子起源和后果，同时深入了解微粒体细胞P450的反应机制。目的1。为了推进我们对cyt b5和cyt P450还原酶如何在cyt P450 2B4催化中发挥其独特作用的理解，我们将与Brian Hoffman博士合作，使用快速冷冻淬火和低温还原EPR实验来捕获和鉴定在cyt P450转换过程中形成的中间体。催化过程中产物的时间依赖性形成将采用快速化学淬火技术进行测量。目标2。由于在野生型cyt P450 2B4和cyt P450还原酶存在下形成的氧化亚铁中间体寿命较短，因此将分离和研究三个突变cyt P450 2B4 (T302A, E301Q, F429H)，它们预计会积累三种不同的较长寿命的反应周期中间体[(Fe+3OOH)- (Fe+ 300)= (Fe+ 200)]，以帮助鉴定和表征瞬时物种。目标3。为了获得cyt b5如何加速cyt P450 2B4有效催化的详细分子理解，将使用定点诱变和双突变周期实验鉴定在cyt P450 2B4和cyt b5结合界面形成蛋白间接触的氨基酸。

DESCRIPTION (provided by applicant): The cytochromes P450 are a ubiquitous superfamily of mixed function oxidases responsible for the oxidation of a vast number of compounds including the majority of drugs used by humans. In the course of our studies to determine how cytochrome b5 (cyt b5) stimulates the metabolism of substrates such as the anesthetic, methoxyflurane, by cytochrome P450 2B4 (cyt P450) we have recently measured, for the first time, the rate of reduction of a microsomal oxyferrous cyt P450, 2B4, by cyt P450 reductase. This experiment led to the discovery that catalysis by cyt P450 2B4 in the presence of cyt P450 reductase proceeds via a reduced oxyferrous intermediate. In striking contrast, catalysis by cyt P450 2134 in the presence of cyt b5 occurs rapidly without formation of a detectable intermediate allowing less time for unproductive side reactions to occur. The tools developed to measure the rate of reduction of oxyferrous cyt P450 2B4 will now be utilized to further probe the catalytic mechanism of cyt P450 and understand how cyt b5 enhances drug metabolism. The short term goals of this proposal are to elucidate the molecular origin and consequences of the strikingly different effects cyt b5 and cyt P450 reductase have on catalysis by cyt P450 2B4 while at the same time, gaining insight into the reaction mechanism of microsomal cyts P450. Aim 1. In order to advance our understanding of how cyt b5 and cyt P450 reductase exert their distinctive effects on cyt P450 2B4 catalysis we will trap and identify the intermediates formed during cyt P450 turnover using rapid freeze quench and cryoreduction EPR experiments in collaboration with Dr. Brian Hoffman. The time dependent formation of product during catalysis will be measured using rapid chemical quench techniques. Aim 2. Since the reduced oxyferrous intermediate which forms in the presence of wild type cyt P450 2B4 and cyt P450 reductase is short lived, three mutant cyts P450 2B4 (T302A, E301Q, F429H) which are expected to accumulate three different longer-lived reaction cycle intermediates [(Fe+3OOH)- (Fe+3OO)= (Fe+2OO)] will be isolated and studied to aid in the identification and characterization of the transient species. Aim 3. In an effort to gain a detailed molecular understanding of how cyt b5 expedites efficient catalysis by cyt P450 2B4, the amino acids forming interprotein contacts at the binding interface between cyt P450 2B4 and cyt b5 will be identified using site-directed mutagenesis and double-mutant cycle experiments.