PLACENTAL 3 BETA-HYDROXYSTEROID DEHYDROGENASE/ISOMERASE

本项目的目的是通过将功能与结构联系起来，确定人类I型3β-羟基类固醇脱氢酶/类固醇5-4-烯-异构酶(3β-HSD/异构酶)的反应机制。在胎盘中，I3型β-HSD/异构酶催化母亲孕烯醇酮转化为孕酮，孕酮是一种在怀孕期间促进子宫平静的激素。该酶还竞争性地利用脱氢表雄酮(胎儿肾上腺近期主要的类固醇产物)来产生雄烯二酮，雄烯二酮进一步代谢为17β-雌二醇。因此，胎盘3β-HSD/异构酶在母亲和胎儿之间架起荷尔蒙交流的桥梁，以调节局部增加的雌激素/孕酮平衡，这与分娩开始有关。I3型β-HSD/异构酶也是乳腺和乳腺肿瘤的主要形式。杆状病毒已经在昆虫细胞中过表达了I型酶的基因。通过亲和标记的多肽研究定位底物结构域。以下研究验证了我们的中心假设：3β-HSD和异构酶活性是在重叠但不同的催化部位催化的。当NAD+被3β-HSD活性还原为NADH时，所产生的NADH通过诱导酶蛋白的构象变化来激活异构酶反应。因此，辅酶在单个区域的利用连接了连续的反应。YXXXK基序中Tyr和Lys的多个突变以及靶向Tyr和Asp残基的多个突变决定了这些氨基酸是否分别催化3β-HSD和异构酶机制。利用3β-HSD位点定向的亲和标记辅因子类似物5‘-(对氟磺酰基[14C]苯甲酰基)腺苷来定位负责NAD+还原的辅因子位点。使用NADH位点定向的类似物8-[(4-bromo-2，3-[3H]dioxobutyl)-thio]-adenosine 5‘-二磷酸来鉴定异构酶激活位点。一项使用两个亲和的烷基化核苷酸的“交叉”研究明确地测试了我们关于单一辅酶结构域的假设。在已确定的辅酶结构域(S)中引入特定的突变，然后杆状病毒在昆虫细胞中表达突变的酶。对纯化突变体的动力学和pH依赖性研究表征了负责底物和辅因子利用的氨基酸，以确定多种反应机制。跨膜结构域的缺失产生了一种完全活性的胞浆酶，无需使用洗涤剂即可纯化，似乎与微粒体野生型酶完全相同。对纯化的胞浆酶和纯化的野生型酶的动力学性质进行了比较。纯胞浆酶制剂中不含洗涤剂，有利于晶体的生长，可用于未来的结构研究。

The objective of this project is to define the reaction mechanisms of human type I 3beta-hydroxysteroid dehydrogenase/steroid 5-4-ene-isomerase (3beta-HSD/isomerase) by relating function to structure. In placenta, type I 3beta-HSD/isomerase catalyzes the conversion of maternal pregnenolone to progesterone, a hormone that promotes uterine quiescence during pregnancy. The enzyme also competitively utilizes dehydroepiandrosterone, the primary steroid product of the fetal adrenal gland near term, to produce androstenedione that is further metabolized to l7beta-estradiol. Thus, placental 3beta-HSD/isomerase bridges hormonal communication between the mother and fetus to mediate the locally increased estrogen/progesterone balance that has been associated with the onset of labor. The type I 3beta-HSD/isomerase is also the dominant form in the mammary gland and breast tumors. The cDNA for the type I enzyme has been over-expressed by baculovirus in insect cells. The substrate domain was localized by affinity radiolabeled peptide studies. The following studies test our central hypothesis: The 3beta-HSD and isomerase activities are catalyzed at overlapping, but distinct, catalytic sites. When NAD+ is reduced to NADH by the 3beta-HSD activity, the NADH produced activates the isomerase reaction by inducing a conformational change in the enzyme protein. Thus, the utilization of coenzyme at a single domain bridges the sequential reactions. Multiple mutations of Tyr and Lys in the YXXXK motifs and of targeted Tyr and Asp residues determine if these amino acids catalyze the 3beta-HSD and isomerase mechanisms, respectively. The cofactor site responsible for NAD+ reduction is localized using the 3beta-HSD-site-directed, affinity labeling cofactor analog, 5'-(p-fluorosulfonyl[14C]benzoyl)adenosine. The isomerase-activator site is identified using the NADH-site-directed analog, 8-[(4-bromo-2,3-[3H]dioxobutyl)-thio]-adenosine 5'-diphosphate. A "cross-over" study using both affinity alkylating nucleotides definitively tests our hypothesis of a single coenzyme domain. Specific mutations are introduced in the identified coenzyme domain(s) followed by baculovirus- expression of the mutant enzymes in insect cells. Kinetic and pH- dependence studies on the purified mutants characterize the amino acids responsible for substrate and cofactor utilization to define the multiple reaction mechanisms. The deletion of a membrane-spanning domain produced a fully active, cytosolic enzyme that can be purified without using detergents and appears to be identical to the microsomal wild-type enzyme. The kinetic properties of the purified cytosolic enzyme are compared to those of the purified wild-type enzyme. The absence of detergent in the pure cytosolic enzyme preparation facilitates the growth of crystals for future structural studies.