BIOCHEMICAL PHARMACOLOGY OF STEROID HORMONES

这项建议的长期目标是澄清 生命系统中类固醇的酶转化机制 通过分离相关酶的纯形式； 它们的分子性质表征及其三维构型 建筑.催化机理的澄清.定义 底物和抑制剂的特异性和立体特异性；以及 活性部位定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一个合乎逻辑的 延长高纯度羟基类固醇的使用范围 脱氢酶我们已经用这些酶开发出了极其敏感的 小样本组织中类固醇分布的分析系统 还有体液。这些研究的动机是需要获得 更好地了解类固醇生物合成和调节 在正常组织和异常生长中的代谢。具体目标 这一建议的主要内容是：1.继续进行研究，旨在阐明 类固醇结合的详细分子结构、形貌 位置、催化机理和极高的催化效率 Al-3-酮类固醇异构酶的联合应用动力学 (包括同位素动力学)、核磁共振、电子顺磁共振、X射线和紫外线 光谱学及其在氨基酸定点诱变中的应用 酸残基可以改变蛋白质的结构和功能。2. 一种激素微量分析综合系统的研制 通过用新的和新的方法分离羟基类固醇脱氢酶 来自能够生长的微生物的严格的特异性 类固醇是它们唯一的碳来源。新基因的克隆和高效表达 这些酶中最有用的将使它们得到更广泛的使用 用于分析目的。直接酶反应器的研究进展 这种羟基类固醇脱氢酶已被固定在固体上 载体，或被困在反胶束中，将扩大 这些分析允许它们直接应用于色谱学 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.