BINDING, BIOSYNTHESIS, AND ACTION OF STEROIDS

本项目将继续研究比较结构和 类固醇结合蛋白的结合部位拓扑学。我们会 利用：(A)亲和标记类固醇(定点定向不可逆 抑制剂)和自杀底物(现场生成的不可逆性 抑制剂)与烷基化残基和多肽的鉴定 (B)一级结构(氨基)的测定 酸序列)，从而使类固醇结合部位(定义在 (A)可局部化和定向，以及(C)X射线结晶学。 对于类固醇相互转换酶，我们还将：(A) 测定氢化物转移到辅因子的立体专一性，(B) 评估活动期类固醇和辅因子的空间关系 以及(C)确定那些靠近氨基酸残基的 催化事件的位置，并可能起到诱导 过渡状态。我们将研究人类胎盘雌二醇17 β-脱氢酶，表观雌二醇17α-和17β- 马胎盘脱氢酶，牛睾丸20α- 羟基类固醇脱氢酶(所有这些都已在 该实验室和可供使用的数量足以 和黄体酮结合球蛋白。 血清由拉里·库恩博士提供。 没有明确的证据表明大自然是如何构建一个 类固醇结合部位或类固醇的催化机制 相互转换酶诱导其上的过渡态 底物。这个项目就是为了回答这些问题。 我们的最终目标是进行X射线结晶学研究 因为我们知道，仅靠这种方法就会给出明确的 关于氨基酸残基在空间上的活性部位的数据 相互关联，并与类固醇和辅因子有关 大分子结合部位。我们还将广泛应用于 亲和力标记类固醇和自杀底物，不那么精确， 但更容易应用的方法是阐明结合位点 结构。我们的基本假设是，当一个人做了足够多的事情 亲和标记化合物和自杀底物的研究 通过随后的X射线结晶学验证，这些更多 容易应用的技术可能会变得足够至少 不同来源类固醇结合部位的共性定义 消息来源。

This project will continue study of the comparative structure and binding site topography of steroid-binding proteins. We will utilize: (a) affinity-labeling steroids (site directed irreversible inhibitors) and suicide substrates (site-generated irreversible inhibitors) with identification of alkylated residues and peptides that contain them, (b) determination of primary structure (amino acid sequence), so that steroid binding sites (peptides defined in (a)) can be localized and oriented, and (c) X-ray crystallography. For the steroid-interconverting enzymes, we will also: (a) determine stereospecificity of hydride transfer to cofactor, (b) evaluate spatial relationships of steroid and cofactor at the active site, and (c) identify those amino acid residues that proximate the site of the catalytic event and presumably play a role in inducing the transition state. We will study human placental estradiol 17 beta-dehydrogenase, the epimeric estradiol 17 alpha-and 17 beta- dehydrogenases from horse placenta, bovine testicular 20 alpha- hydroxysteroid dehydrogenase (all of which have been purified in this lab and are available in quantities sufficient to carry out these studies) and progesterone binding globulin of guinea pig serum supplied by Dr. Larry Kuhn. No unequivocal evidence exists as to how nature constructs a steroid binding site or the catalytic mechanism by which a steroid interconverting enzyme induces the transition state on its substrate. This project is designed to answer these questions. Our ultimate goal is to carry out X-ray crystallographic studies because we know that this method alone will give unequivocal data as to how active site amino acid residues are spatially related to each other and to the steroid and cofactor at the macromolecular binding site. We will also make wide application of affinity-labeling steroids and suicide substrates, less precise, but more readily applicable methods for elucidating binding site structure. Our basic hypothesis is that, after one has done enough studies with affinity-labeling compounds and suicide substrates with subsequent validation by X-ray crystallogaphy, these more readily applicable techniques may become adequate for at least definition of commonality in steroid binding sites from various sources.