RATE LIMITING STEPS IN CYTOCHROME P450 CATALYSIS

细胞色素 P450 催化中的限速步骤

• 批准号: 2767941
• 负责人: GROVER P MILLER
• 金额: $ 3.17万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-15 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2767941
• 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在细胞过程中的作用使得对P450机制的理解成为研究的热点。催化机制的知识是阐明酶特异性的一个组成部分，酶特异性是药物设计、生物修复和癌症风险评估等领域的一个重要课题。虽然证据通常支持描述细胞色素P450介导的底物氧化的替代机制途径，但仍不清楚是什么真正决定了产物形成的速率，从而决定了氧化途径。为了解决这个问题，我们建议采用由人P450 1A 2和各种苯甲醚和N，N-二甲基苯胺衍生物组成的模型系统，并确定包括这些底物的去甲基化的催化步骤。该系统的设计旨在通过使氧化产物最小化来简化感兴趣的反应，并且能够采用各种技术分析催化循环的步骤。作为这项工作的补充，将在涉及替代氧化途径的关键区域进行定点诱变。所得突变体将根据适当的模型催化机制进行表征和分析。