Participation of Cytochrome b5 in Anesthetic Metabolism

描述（由申请人提供）：细胞色素P450是一种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。在我们的研究过程中，为了确定细胞色素b5（cyt b5）如何通过细胞色素P450 2B 4（cyt P450）刺激麻醉剂甲氧氟烷等底物的代谢，我们最近首次测量了微粒体氧化铁细胞色素P450的还原率，2B 4，由cyt P450还原酶。该实验发现，在cyt P450还原酶存在下，cyt P450 2B 4的催化作用通过还原的含氧亚铁中间体进行。与此形成鲜明对比的是，在细胞色素b5存在下，细胞色素P450 2134的催化作用迅速发生，而不会形成可检测的中间体，从而减少了发生非生产性副反应的时间。开发用于测量氧化亚铁细胞色素P450 2B 4还原速率的工具现在将用于进一步探测细胞色素P450的催化机制，并了解细胞色素B5如何增强药物代谢。该提案的短期目标是阐明细胞色素B5和细胞色素P450还原酶对细胞色素P450 2B 4催化作用的显著不同效应的分子起源和后果，同时深入了解微粒体细胞色素P450的反应机制。目标1.为了进一步了解cyt b5和cyt P450还原酶如何对cyt P450 2B 4催化发挥其独特作用，我们将与Brian霍夫曼博士合作，使用快速冷冻淬火和冷冻还原EPR实验捕获并鉴定cyt P450周转期间形成的中间体。将使用快速化学淬灭技术测量催化过程中产物形成的时间依赖性。目标2.由于在野生型cyt P450 2B 4和cyt P450还原酶存在下形成的还原型含氧亚铁中间体是短寿命的，因此三个突变型cyt P450 2B 4（T302A，E301Q，F429 H），其预期积累三种不同的长寿命反应循环中间体[（Fe+3 OOH）-（Fe +3 OO）=（Fe+2 OO）]将进行分离和研究，以帮助识别和表征瞬时物质。目标3。在努力获得一个详细的分子理解细胞色素b5如何加速有效的催化细胞色素P450 2B 4，氨基酸形成蛋白质间的接触在细胞色素P450 2B 4和细胞色素b5之间的结合界面将被确定使用定点诱变和双突变体循环实验。

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.