BINDING, BIOSYNTHESIS, AND ACTION OF STEROIDS

拟议的项目旨在继续研究 各种高亲和力的结构和结合部位的形貌 类固醇结合蛋白。我们将利用：a)亲和标记类固醇 (位点定向的不可逆抑制剂)，随后鉴定 烷基化残基和含有它们的多肽的测序，b) 一级结构的测定，以及c)x射线结晶学。在……里面 此外，在这些蛋白质是酶的情况下，我们将：a)确定 氢转移到辅因子的立体专一性，b)评估空间 类固醇与辅因子可逆性变化的关系 活性部位的结合步骤，以及c)鉴定那些 在催化事件的位置附近，并可能在 诱导过渡状态。类固醇脱氢酶将被用作模型 包括人胎盘雌二醇17β-脱氢酶、马胎盘 表观脱氢酶(雌二醇17α和17β脱氢酶)，大鼠 猪或牛卵巢20α-脱氢酶和20-脱氢酶 性腺。此外，鸡输卵管的亲和标记研究 黄体酮受体和可能的小牛子宫雌二醇受体 搞定了。目前，还没有真正的证据表明进化是如何发生的 该过程构建了一个类固醇结合部位。结构研究，类似 本项目中提议的项目已与 2-羟基酸脱氢酶，并提供了对 与催化事件相关的结合部位结构和商业化。 他们的努力是值得的。我们认为，在以下方面也是如此 结合和催化涉及类固醇的反应的大分子。

The proposed project is designed to continue study of the comparative structure and binding site topography of various high-affinity steroid-binding proteins. We will utilize: a) affinity-labeling steroids (site-directed irreversible inhibitors) with subsequent identification of alkylated residues and sequencing of the peptides that contain them, b) determination of primary structure, and c) x-ray crystallography. In addition, where these proteins are enzymes, we will: a) determine stereospecificity of hydrogen transfer to cofactor, b) evaluate spatial relationships of steroid and cofactor as they undergo the reversible binding step at the active site, and c) identify those residues that proximate the site of the catalytic event and presumably play a role in inducing the transition state. Steroid dehydrogenases to be used as models include human placental estradiol 17Beta-dehydrogenase, the horse placental epimeric dehydrogenases (estradiol 17Alpha- and 17Beta- dehydrogenase), rat ovarian 20Alpha-dehydrogenase and a 20-dehydrogenase from pig or cow gonad. In addition, affinity-labeling studies of chicken oviduct progesterone receptor and possibly, calf uterine estradiol receptor will be done. Currently, no real evidence exists as to how the evolutionary process has structured a steroid binding site. Structural studies, similar to those proposed in this project have been carried out with the 2-hydroxyacid dehydrogenases and have provided "in depth" understanding of binding site structure and merchanisms pertinent to the catalytic event. They have been worth the effort. We submit that the same is true in terms of those macromolecules that bind and catalyze reactions involving steroids.