BINDING, BIOSYNTHESIS, AND ACTION OF STEROIDS

本项目将继续研究比较结构， 类固醇结合蛋白的结合位点形貌。 我们将 利用：（a）亲和标记类固醇（位点定向不可逆 抑制剂）和自杀底物（位点产生的不可逆 抑制剂），并鉴定烷基化残基和肽 （B）测定一级结构（氨基 酸序列），使得类固醇结合位点（在 (a))可以定位和定向，以及（c）X射线晶体学。 对于类固醇相互转化酶，我们还将：（a） 确定氢化物转移至辅因子的立体特异性，（B） 评价类固醇和辅因子在活动时的空间关系 位点，和（c）鉴定接近所述位点的那些氨基酸残基。 催化事件的位点，并可能在诱导 过渡状态。 我们将研究人胎盘雌二醇17 β-脱氢酶，差向异构雌二醇17 α-和17 β- 马胎盘、牛睾丸20 α- 羟基类固醇脱氢酶（所有这些都已在 本实验室提供的数量足以进行 这些研究）和豚鼠孕酮结合球蛋白 拉里·库恩医生提供的血清 没有明确的证据表明大自然是如何构造 类固醇结合位点或类固醇结合位点的催化机制 相互转化酶诱导其上的过渡态 衬底 本项目旨在回答这些问题。 我们的最终目标是进行X射线晶体学研究 因为我们知道，单凭这种方法， 关于活性位点氨基酸残基如何在空间上 与类固醇和辅因子相关， 大分子结合位点 我们还将广泛应用 亲和标记类固醇和自杀底物，不太精确， 但更容易应用的阐明结合位点的方法 结构 我们的基本假设是，当一个人做了足够多的事情后， 亲和标记化合物和自杀底物的研究 随后通过X射线晶体学验证，这些更多 容易应用的技术可能足以至少 不同来源的类固醇结合位点的共性定义 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.