STUDIES ON THE ACTIVE SITES AND MECHANISMS OF CYTOCHROME P450

细胞色素P450活性位点及机制研究

• 批准号: 3838460
• 负责人: K KORZEKWA
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3838460
• 关键词: active sites; chemical binding; chemical kinetics; chimeric proteins; conformation; cytochrome P450; electron transport; enzyme mechanism; enzyme model; enzyme substrate; human tissue; model design /development; mutant; oxidation; steroid hormone metabolism; stoichiometry; testosterone

The cytochrome P450s are a superfamily of monooxygenases involved in the metabolism of both exogenous and endogenous compounds. Although there has been a tremendous effort to determine the details of the catalytic cycle, several aspects of these enzymes have hindered their complete characterization. These include the absence of crystal structures (for mammalian enzymes) the complexity of the catalytic cycle, presence of multiple isozymes, the diversity of substrates (for some isozymes), and the versatility of the active oxygen species. While many of the mechanisms of the actual substrate oxidation steps have been defined, the aspects of these enzymes responsible for substrate specificity and catalytic efficiency are still unknown. The goal of this research is to explore the mechanisms of oxygen activation, substrate oxidation and the topology of 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 have performed full kinetic analyses, including isotope effect experiments on wild type and mutant P450s. Stoichiometry experiments to characterize the effect of the mutations on the enzyme mechanisms are in progress.

细胞色素P450是单加氧酶的超家族，参与 外源和内源化合物的代谢。尽管有 为确定催化循环的细节付出了巨大的努力， 这些酶的几个方面阻碍了它们的完全 人物刻画。这些问题包括没有晶体结构(例如 哺乳动物酶)催化循环的复杂性，存在 多个同工酶，底物的多样性(对于某些同工酶)，以及 活性氧物种的多功能性。虽然许多人 已经定义了实际衬底氧化步骤的机制， 这些酶负责底物专一性和 催化效率仍不得而知。这项研究的目标是 探讨了氧活化、底物氧化和氧化的机理。 P450活性部位的拓扑结构。项目中使用的方法包括 重组DNA技术，酶和同位素效应的测定 野生型和突变型酶的动力学和动力学分析。在……里面 过去，我们已经推导出了几个综合动力学方程 描述观察到的细胞色素P450动力学同位素效应的模型 催化氧化作用。这些模型表明，观测到的同位素 效应可以提供有关结合构象和 导致水形成的未耦合电子通量的量。 关于睾酮代谢的先前研究由几个 表达的P450同工酶及其嵌合和突变形式(由 Frank Gonzalez博士)透露，几个氨基酸残基的修饰 处于危险位置的人会显著影响代谢物的模式。我们 进行了全面的动力学分析，包括同位素效应实验 野生型和突变型P450。化学计量学实验来表征 突变对酶机制的影响正在进行中。