STUDIES ON THE ACTIVE SITES OF CYTOCHROMES P-450

细胞色素 P-450 活性位点的研究

• 批准号: 3878913
• 负责人: K KORZEKWA
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 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/3878913
• 关键词: chemical binding; chemical reaction; cytochrome P450; drug metabolism; enzyme mechanism; enzyme model; enzyme structure; enzyme substrate; enzyme substrate complex; genetic manipulation; isozymes; radionuclides; testosterone; unspecific monooxygenase

The cytochrome P-450s 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 exogenous chemical compounds. The first is the generally broad substrate and regio-specificity 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.These characteristics are being explored with the ultimate goal of predicting how changes in composition and structure of drugs will alter metabolic pathways. While most cytochrome P-450s have low substrate specificity, isozymes used for the metabolism of endogenous substance can be very specific. An example of a high specificity isozyme is aromatase, the enzyme responsible for the conversion of androgens to estrogens. This project describes our attempts at defining the binding influences responsible for certain drug metabolizing P-450 isozymes and the electronic and protein interactions responsible for the unusual mechanism of the third oxidation of aromatase. Methods used in the project include recombinant DNA techniques, determination of enzyme kinetics, and molecular modelling techniques. We have studied the metabolism of testosterone by clones, chimeras and single point mutants of P-450IIA1, IIA2 and P-450b using expressed P-450s provided by Dr. Frank Gonzales and his associates (LMC,NCI) . These studies have revealed that 1) modification of a few amino acid residues in critical positions can markedly affect not only the turnover number and pattern of metabolites, but also the enzyme stability, 2) regions of the polypeptide involved in binding are different for different families of isozymes and 3) the tertiary structure of P-450cam may be a valid model for mammalian P-450s.

细胞色素P-450是一个能够氧化多种细胞色素的同工酶家族。 各种内源性和外源性化合物。的两个特征 这些酶使得有限数量的同工酶能够 代谢大量不同的外源化合物。的 首先是一般广泛的基板和区域特异性大概是由于 相对非特异性的底物结合特性和多种 结合方向第二种是多功能的活性氧化物种 它能够氧化各种官能团。这些 我们正在探索这些特征，最终目标是预测 药物组成和结构的变化将改变代谢 途径。虽然大多数细胞色素P-450具有低底物特异性， 用于内源性物质代谢的同工酶可以非常 特定.高特异性同工酶的一个例子是芳香酶， 负责将雄激素转化为雌激素。这个项目 描述了我们试图定义负责的约束影响， 某些药物代谢P-450同工酶和电子蛋白质 负责第三氧化的不寻常机制的相互作用 芳香化酶该项目使用的方法包括重组DNA 技术、酶动力学测定和分子建模 技术.我们研究了克隆人睾丸激素的代谢， 使用本发明的嵌合体和P-450 IIA 1、IIA 2和P-450 b的单点突变体， 弗兰克冈萨雷斯博士和他的同事提供的P-450 (LMC，NCI）。这些研究表明：1）一些氨基的修饰 关键位置上的酸残基不仅可以显著影响 代谢物的周转数和模式，还有酶的稳定性， 2)参与结合的多肽的区域对于 3）P-450 cam的三级结构 可能是哺乳动物P-450的有效模型。