P450BM-3--MECHANISM OF ELECTRON TRANSFER 02 ACTIVATION

P450BM-3--电子转移02激活机制

• 批准号: 3302516
• 负责人: JULIAN A PETERSON
• 金额: $ 14.55万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3302516
• 关键词: Bacillus megaterium; NADPH cytochrome c2 reductase; X ray crystallography; antibody formation; chimeric proteins; crosslink; cytochrome P450; cytochrome b5 reductase; electron transport; enzyme complex; enzyme mechanism; enzyme structure; flavoproteins; hemoprotein structure; laboratory rabbit; microsomes; oxidation; site directed mutagenesis; thermodynamics; toxin metabolism

The long range goal of this research project is to elucidate the mechanism of electron transfer and substrate and oxygen activation by cytochrome P- 450-type enzyme systems. P-450 enzymes systems can be divided into two general classes; 1) those which are have only two protein components in their electron transfer system: an NADPH-P-450 reductase and the P-450; and, 2) those which have three proteins in their electron transfer system: a flavoprotein-iron sulfur protein reductase, an iron sulfur protein ,and the P-450 is relatively specific. In eucaryotes, these enzymes are found in the microsomal and mitochondria fractions respectively. While much is known about protein/protein interaction in the mitochondrial-type system, as a consequence of the vast amount of work which has been done with both P-450cam and P-450scc, there is relatively little precise structural information regarding the very important microsomal drug metabolizing enzymes because of the lack of a comparable soluble enzyme on which to conduct model studies. The recent isolation of a soluble P-450 (P-450BM-3) from Bacillus megaterium containing both the P-450 reductase and the P-450 on a single polypeptide chain and the establishment of the similarities between this enzyme and mammalian microsomal P-450 systems was very exciting. This protein should serve as the model system to clarify structural and mechanistic questions regarding these important drug metabolizing enzymes. With the recent cloning and expression of P-450BM-3, this enzymes is now available in gram quantities in homogeneous form. The Specific Aims of this project are to determine: 1) the kinetics and thermodynamics of the reactions of the individual redox active centers of this multidomain protein; 2) the factors which control protein/protein interaction and domain recognition during electron transfer and oxygen activation; and, 3) the three-dimensional structure of this protein using x-ray crystallography. The first phase will divide (physically and kinetically) the domains of the protein into the reductase and P-450 regions and compare the properties of these domains to the respective microsomal proteins using appropriate biophysical techniques. The second phase will focus on the control of electron flux between the reductase and P-450 domains. The techniques used will complement those employed in phase one but will also include chemical modification, chemical cross-linking and site-directed mutagenesis to identify the residues and regions important in electron transfer and protein/protein recognition. The third phase, which will be conducted concurrently with the other two, is the determination of the three-dimensional structure of the holoenzyme and the individual domains. The results obtained will enable us to better understand the factors which control the oxidation of fatty acids, drugs, carcinogens, steroids, and xenobiotics by this class of P-450 enzymes

本研究项目的长期目标是阐明 电子转移和底物和氧活化的细胞色素P- 450-型酶系统。 P-450酶系可分为两类 一般类别; 1）那些只有两种蛋白质成分， 它们的电子传递系统：NADPH-P-450还原酶和P-450; 和，2）在其电子传递系统中具有三种蛋白质的那些： 黄素蛋白-铁硫蛋白还原酶，铁硫蛋白，和 P-450是相对具体的。 在真核生物中， 分别在微粒体和线粒体组分中。 虽然很多 已知蛋白质/蛋白质相互作用在蛋白质型系统中， 由于大量的工作已经完成， P-450 cam与P-450 SCC相比，精密结构相对较少 关于非常重要的微粒体药物代谢 酶，因为缺乏可比较的可溶性酶， 进行模型研究。 芽孢杆菌可溶性P-450（P-450 BM-3）的分离纯化 在一个单一的含有P-450还原酶和P-450的巨大的 多肽链和建立之间的相似性， 酶和哺乳动物微粒体P-450系统是非常令人兴奋的。 这 蛋白质应该作为模型系统，以澄清结构和 关于这些重要的药物代谢酶的机制问题。 随着最近P-450 BM-3的克隆和表达，这种酶现在被 以克为单位以均质形式提供。 的具体目标 本项目的主要目的是确定：1）动力学和热力学的， 该多畴的各个氧化还原活性中心的反应 蛋白质; 2）控制蛋白质/蛋白质相互作用的因素， 在电子转移和氧活化期间的结构域识别;以及，3） 用X射线扫描这种蛋白质的三维结构 结晶学 第一阶段将分裂（物理和动力学） 将蛋白质的结构域插入还原酶和P-450区域，并比较 这些结构域对各自微粒体蛋白的性质， 适当的生物物理技术。 第二阶段将侧重于 控制还原酶和P-450结构域之间的电子通量。 的 所使用的技术将补充第一阶段所使用的技术，但也将 包括化学修饰、化学交联和定点 诱变以鉴定在电子传递中重要的残基和区域， 转移和蛋白质/蛋白质识别。 第三阶段将是 与其他两个同时进行，是确定 全酶和各个结构域的三维结构。 所获得的结果将使我们能够更好地了解 控制脂肪酸、药物、致癌物质、类固醇的氧化， 通过这类P-450酶的异生物质