PLACENTAL 3B-HYDROXYSTEROID DEHYDROGENASE ISOMERASE

该项目的目标是确定反应机理 人胎盘3β-羟基类固醇脱氢酶/类固醇5-和4-烯- 异构酶(3β-HSD/异构酶)通过将功能与结构联系起来。在……里面 胎盘，3β-HSD/异构酶催化母体 孕烯醇酮到孕酮，一种促进子宫平静的激素 在怀孕期间。这种酶竞争性地利用 脱氢表雄酮，胎儿肾上腺的主要类固醇产物 腺体，产生雄烯二酮，进一步代谢到 17β-雌二醇。因此，胎盘3β-HSD/异构酶连接荷尔蒙 母亲和胎儿之间的沟通，以调解当地的 雌激素/孕激素平衡增加与 分娩开始了。3β-HSD/异构酶的特性可能最终 允许对胎盘酶进行药物控制，不依赖于 不同的性腺/肾上腺同工酶，防止早产。 从人胎盘中提纯的同质酶在手中。野生型 杆状病毒在昆虫细胞中过表达的酶，并发现 与天然胎盘酶在动力学上是相同的。底物的顺序 并研究了辅酶结合对3β-HSD和异构酶活性的影响。 利用经典的同位素配体交换和新的亲和力 标记/配体保护实验。胎盘异构酶反应 与已知的基本辅因子的作用机制和激活机制进行了比较 细菌异构酶机制(不需要辅因子) 19-去甲睾酮和17-β-雌二醇结合时的光谱变化 在NADH存在或不存在的情况下对酶的作用。停流 光谱学实验解决了我们的假设，即依赖于时间的 构象变化介导了NADH诱导的异构酶激活。 亲和标记和配体保护实验绘制了结合图 3β-HSD底物、异构酶底物和辅因子在 已知的这种单一的多功能蛋白质的一级结构。vbl.使用 我们的编码胎盘3β-HSD/异构酶的cDNA，可能是催化的 这些识别区域中的氨基酸被用修饰的 人工合成的寡核苷酸。可能的辅因子与膜锚定 区域将被删除。突变的和野生型的cdna通过 昆虫SF-9细胞悬液中的杆状病毒。功能界别 每一种表达的、纯化的突变酶的意义由 之前对天然酶进行了严格的动力学分析。这些 分析包括测量3β的米氏常数。 HSD底物、异构酶底物和辅因子，抑制动力学 对于产品类固醇和NADH，以及使用 亲和烷化剂，专用于修饰的结合部位。这些 结构/功能研究定位催化氨基酸 一级结构中的酶活性，并验证我们独特的假设 顺序的3-β-HSD和异构酶反应是在 在单个类固醇结合区中的邻接位置。

The objective of this project is to define the reaction mechanisms of human placental 3beta-hydroxysteroid dehydrogenase/steroid 5->4-ene- isomerase (3beta-HSD/isomerase) by relating function to structure. In placenta, 3beta-HSD/isomerase catalyzes the conversion of maternal pregnenolone to progesterone, a hormone that promotes uterine quiescence during pregnancy. The enzyme competitively utilizes dehydroepiandrosterone, the primary steroid product of the fetal adrenal gland near term, to produce androstenedione that is further metabolized to 17beta-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. Characterization of 3beta-HSD/isomerase may ultimately allow pharmacologic control of the placenta enzyme, independent of the different gonadal/adrenal isoenzyme, to prevent premature births. Homogeneous enzyme purified from human placenta is in-hand. Wild-type enzyme as been overexpressed by baculovirus in insect cells and found to be kinetically identical to native placenta enzyme. The order of substrate and coenzyme binding for the 3beta-HSD and isomerase activities is studied using both classic isotopic ligand exchange and novel affinity labeling/ligand protection experiments. The placenta isomerase reaction mechanism and activation by essential cofactor are compared to the known bacterial isomerase mechanism (with no cofactor requirement) by measuring spectral changes in 19-nortestosterone and 17beta-estradiol upon binding to the enzyme in the presence or absence of NADH. Stopped-flow spectroscopy experiments address our hypothesis that a time-dependent conformational change mediates the NADH-induced activation of isomerase. Affinity radiolabeling and ligand protection experiments map the binding sites for 3beta-HSD substrate, isomerase substrate, and cofactor in the known primary structure of this single, multifunctional protein. Using our cDNA that encodes placental 3beta-HSD/isomerase, probable catalytic amino acids in these identified regions are mutated using modified synthetic oligonucleotides. Probable cofactor and membrane anchoring regions are deleted. The mutated and wild-type cDNA are over-expressed by baculovirus in suspensions of insect Sf-9 cells. The functional significance of each expressed, purified mutant enzyme is determined by rigorous kinetic analyses previously applied to the native enzyme. These analyses include the measurement of Michaelis-Menton constants for 3beta- HSD substrates, isomerase substrates, and cofactors, inhibition kinetics for product steroids and NADH, and inactivation/protection profiles using affinity alkylators that are specific for the modified binding site. These structure/function studies localize the catalytic amino acids for the enzyme activities in the primary structure and test our unique hypothesis that the sequential 3beta-HSD and isomerase reactions are catalyzed at contiguous sites in a single steroid binding region.