PLACENTAL 3B-HYDROXYSTEROID DEHYDROGENASE ISOMERASE

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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.