STUDIES ON THE ACTIVE SITES AND MECHANISMS OF CYTOCHROME P450

细胞色素P450是一个同工酶家族，能够广泛地氧化 内源和外源化合物的种类。两个特点 使有限数量的同工酶有可能 代谢大量不同种类的化合物。第一个是 一般广泛的底物和区域特异性可能是由于 相对非特异的底物结合特性和多个 绑定方向。第二种是一种多功能的活性加氧剂 能够氧化各种官能团的物种。这个 本研究的目的是探索氧激活的机制， 底物氧化和P450活性中心的拓扑结构。方法 在该项目中使用的包括重组DNA技术，测定 酶和同位素效应动力学，并对两者进行动力学分析 类型和突变的酶。在过去，我们推导出了几个方程 用于描述观察到的动力学同位素的综合动力学模型 对细胞色素P450催化氧化的影响。这些模型表明 观察到的同位素效应可以提供关于这两种结合的信息 构象和未耦合电子通量的量导致 水层。睾丸素代谢的研究进展 几种表达的P450同工酶及其嵌合体和突变体 Forms(由Frank Gonzalez博士开发)显示，对 关键位置上很少的氨基酸残基能显著影响 代谢物的模式。我们现在正在进行全面的表演 动力学分析，包括同位素效应实验和化学计量 野生型酶特性的实验及其对 酶机制上的突变。

The cytochromes P450 are a family of isozymes capable of oxidizing a wide variety of both endogenous and exogenous compounds. Two characteristics of these enzymes make it possible for a limited number of isozymes to metabolize a vast and varied array of chemical compounds. The first is the generally broad substrate and regiospecificity presumably due to relatively nonspecific substrate binding characteristics and multiple binding orientations. The second is a versatile active oxygenating species that is capable of oxidizing a variety of functional groups. The goal of this research is to explore the mechanisms of oxygen activation, substrate oxidation and the topology of the P450 active sites. Methods used in the project include recombinant DNA techniques, determination of enzyme and isotope effect kinetics, and kinetic analysis of both wild type and mutant enzymes. In the past, we have derived several equations for comprehensive kinetic models to describe the observed kinetic isotope effects on cytochrome P450 catalyzed oxidations. These models suggest that the observed isotope effects can provide information on both binding conformations and the amount of uncoupled electron flux that results in water formation. Previous studies on the metabolism of testosterone by several of the expressed P450 isozymes and their chimeric and mutant forms (developed by Dr. Frank Gonzalez) revealed that modification of a few amino acid residues in critical positions can markedly affect the pattern of metabolites. We are now in the process of performing full kinetic analyses, including isotope effect experiments, and stoichiometry experiments to characterize wild-type enzymes and the effect of the mutations on the enzyme mechanisms.