RATE LIMITING STEPS IN CYTOCHROME P450 CATALYSIS

细胞色素 P450 催化中的限速步骤

• 批准号: 6138315
• 负责人: GROVER P MILLER
• 金额: $ 3.75万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6138315
• 关键词: chemical kinetics; cytochrome P450; enzyme activity; enzyme mechanism; enzyme model; enzyme substrate; radiotracer; site directed mutagenesis; stop flow technique

The role of P450-mediated reactions in cellular processes has made the understanding of P450 mechanism a focus of intense research. The knowledge of catalytic mechanisms is an integral part of the elucidation o enzyme specificity, an important topic in areas as diverse as drug design, bioremediation, and cancer risk estimation. Whereas evidence generally supports alternate mechanistic pathways describing Cytochrome P450-mediated substrate oxidation, it is still unclear what really determines the rate of product formation and hence the pathway of oxidation. To address this issue, we propose to employ a model system consisting of human P450 1A2 and various anisole and N,N-dimethylaniline derivatives and to determine the catalytic steps comprising the demethylation of these substrates. The design of this system is intended to simplify the reactions of interest by minimizing the products of oxidation and to enable an analysis of the steps of the catalytic cycle employing a variety of techniques. As a complement to this work, site- directed mutagenesis will be carried out at key regions implicated in alternate oxidative pathways. The resulting mutants will be characterized and analyzed in terms of the appropriate model catalytic mechanisms.

P450介导的反应在细胞过程中的作用使对P450机制的理解成为激烈研究的重点。催化机制的知识是阐明O酶特异性不可或缺的一部分，这是像药物设计，生物修复和癌症风险估计等多样化领域的重要主题。尽管证据通常支持描述细胞色素P450介导的底物氧化的替代机械途径，但仍不清楚是什么真正决定了产物形成的速率以及因此氧化途径。为了解决这个问题，我们建议采用一个模型系统，该模型系统由人类P450 1A2和各种苯甲酸盐和N，N-二甲基苯胺衍生物组成，并确定包括这些底物脱甲基化的催化步骤。该系统的设计旨在通过最大程度地减少氧化产物并对采用各种技术进行催化循环的步骤进行分析来简化感兴趣的反应。作为对这项工作的补充，将在与替代氧化途径有关的关键区域进行定位的诱变。将根据适当的模型催化机制来表征和分析所得突变体。