RATE LIMITING STEPS IN CYTOCHROME P450 CATALYSIS

P450介导的反应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。对催化机制的了解是阐明酶特异性的一个重要组成部分，在药物设计、生物修复和癌症风险评估等领域都是一个重要的课题。虽然证据普遍支持描述细胞色素P450介导的底物氧化的替代机制途径，但仍然不清楚是什么真正决定了产物形成的速度，从而决定了氧化途径。为了解决这个问题，我们建议使用一个由人P450 1A2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去甲基化的催化步骤。该系统的设计意在通过最大限度地减少氧化产物来简化感兴趣的反应，并利用各种技术分析催化循环的各个步骤。作为这项工作的补充，定点突变将在替代氧化途径中涉及的关键区域进行。由此产生的突变体将根据适当的模型催化机理进行表征和分析。

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.