P450BM-3--MECHANISM OF ELECTRON TRANSFER 02 ACTIVATION

这一研究项目的长期目标是阐明其作用机制。 细胞色素P-2对电子传递、底物和氧活化的影响 450型酶系统。P-450酶系统可分为两个 一般类；1)那些只有两个蛋白质成分的 它们的电子传递系统：NADPH-P-450还原酶和P-450； 以及，2)电子转移系统中有三种蛋白质的那些： 黄素蛋白-铁硫蛋白还原酶，铁硫蛋白，以及 P-450是相对特定的。在真核生物中，这些酶被发现 分别在微粒体组分和线粒体组分中。虽然很多都是 已知线粒体类型系统中的蛋白质/蛋白质相互作用， 作为对两者进行的大量工作的结果 P-450凸轮和P-450scc，精密结构相对较少 关于非常重要的微生物体药物代谢的信息 酶，因为缺乏一种可与之媲美的可溶性酶 开展模型研究。 芽孢杆菌中可溶性P-450(P-450BM-3)的最新分离 同时含有P-450还原酶和P-450的巨噬细胞 多肽链与建立这两者之间的相似性 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来阐明结构和 关于这些重要的药物代谢酶的机制问题。 随着最近P-450BM-3的克隆和表达，这种酶现在已经 有均质形式的克量的。的具体目标 这个项目是为了确定：1)动力学和热力学 该多结构域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； 电子转移和氧活化过程中的结构域识别；3) 用X-射线研究该蛋白质的三维结构 结晶学。第一阶段将(物理和运动学)分开 将该蛋白的结构域分为还原酶和P-450区，并进行比较 这些结构域对相应的微粒体蛋白的特性 适当的生物物理技术。第二阶段将重点放在 还原酶和P-450结构域之间电子流量的控制。这个 使用的技术将补充第一阶段使用的技术，但也将 包括化学修饰、化学交联和位点定向 利用诱变技术鉴定电子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的是确定 全酶和单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、类固醇和 这类P-450酶产生的外源物质

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