BIOCHEMICAL PHARMACOLOGY OF STEROID HORMONES

这项建议的长远目标是阐明 甾体在生命系统中的酶促转化机制 通过分离纯形式的相关酶; 表征其分子特性和三维 体系结构;催化机理的阐明;催化剂的定义 底物和抑制剂特异性和立体特异性;和 活性位点导向和基于机理的特异性抑制剂的设计 干扰或抑制类固醇的生物合成和代谢。作为逻辑 高纯度羟基类固醇的可利用性的扩展 我们已经用这些酶来开发非常敏感的 用于分析小样本组织中的类固醇分布的系统 和体液这些研究的动机是需要获得 更好地了解类固醇生物合成的调节， 在正常组织和异常生长中的代谢。具体目标 该建议的主要内容是：1.继续进行旨在阐明 详细的分子结构，类固醇结合的地形 催化剂的活性部位、催化机理和显着的催化效率 α 1 - 3-酮甾体异构酶通过动力学的组合使用 （包括同位素动力学）、NMR、EPR、X射线和紫外线 氨基酸定点突变的应用 酸残基修饰蛋白质的结构和功能。2. 类固醇微量分析综合系统的研制 通过分离羟基类固醇脱氢酶， 微生物的严格特异性， 类固醇作为它们唯一的碳源。的克隆和过表达 这些酶中最有用的一种将使它们更广泛地获得 用于分析目的。直接酶反应器的发展， 这种羟基类固醇脱氢酶已经固定在固体上， 支持，或截留在反胶束，将扩大范围， 这些分析通过允许它们直接应用于色谱分析， 含有有机溶剂的馏分。

The long-term objectives of this proposal are to elucidate the mechanisms of enzymatic transformations of steroids in living systems through the isolation in pure form of the pertinent enzymes; characterization of their molecular properties and three-dimensional architecture; clarification of catalytic mechanisms; definition of substrate and inhibitor specificities and stereospecificities; and design of active-site-directed and mechanism-based specific inhibitors to perturb or restrain steroid biosynthesis and metabolism. As a logical extension of the availability of highly purified hydroxysteroid dehydrogenases we have used these enzymes to develop extremely sensitive systems for the analysis of steroid profiles in small samples of tissues and body fluids. These studies are motivated by the need to obtain better understanding of the regulation of steroid biosynthesis and metabolism in normal tissues and in abnormal growth. The specific aims of this proposal are: 1. Continuation of studies designed to elucidate the detailed molecular structure, topography of the steroid-binding site, catalytic mechanism, and remarkably high catalytic efficiency of al-3-ketosteroid isomerase through the combined use of kinetic (including isotope-kinetic), NMR, EPR, X-ray and ultraviolet spectroscopy, and the application of site-specific mutagenesis of amino acid residues to modify the structure and function of the protein. 2. Development of a comprehensive system for the microanalysis of steroids through the isolation of hydroxysteroid dehydrogenases with novel and stringent specificities from microorganisms capable of growing on steroidi as their only source of carbon. Cloning and over-expression of the most useful of these enzymes will make them more widely available for analytical purposes. Development of direct enzyme reactors in which such hydroxysteroid dehydrogenases have been immobilized on solid supports, or entrapped in reverse micelles, will extend the scope of these analyses by permitting their direct application to chromatographic fractions containing organic solvents.