Human placental 3β-hydroxy-5-ene-steroid dehydrogenase and steroid 5 → 4-ene-isomerase: Purification from microsomes, substrate kinetics, and inhibition by product steroids

Abstract

In human pregnancy, placental 3β-hydroxy-5-ene-steroid dehydrogenase and steroid 5 → 4-ene-isomerase produce progesterone from pregnenolone and metabolize fetal dehydroepiandrosterone sulfate to androstenedione, an estrogen precursor. The enzyme complex was solubilized from human placental microsomes using the anionic detergent, sodium cholate. Purification (500-fold, 3.9% yield) was achieved by ion exchange chromatography (Fractogel-TSK DEAE 650-S) followed by hydroxylapatite chromatography (Bio-Gel HT). The purified enzyme was detected as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis (monomeric M r = 19,000). Fractionation by gel filtration chromatography at constant specific enzyme activity supported enzyme homogeneity and determined the molecular mass (M r = 76,000). The dehydrogenase and isomerase activities copurified. Kinetic constants were determined at pH 7.4, 37°C for the oxidation of pregnenolone ( K m = 1.9 μM, V max = 32.6 nmol/min/mg) and dehydroepiandrosterone ( K m = 2.8μM, V max = 32.0 nmol/min/mg) and for the isomerization of 5-pregnene-3,20-dione ( K m = 9.7 μM, V max = 618.3 nmol/min/mg) and 5-androstene-3,17-dione ( K m = 23.7μM, V max = 625.7 nmol/min/mg). Mixed substrate analyses showed that the dehydrogenase and isomerase reactions use the appropriate pregnene and androstene steroids as alternative, competitive substrates. Dixon analyses demonstrated competitive inhibition of the oxidation of pregnenolone and dehydroepiandrosterone by both product steroids, progesterone and androstenedione. The enzyme has a 3-fold higher affinity for androstenedione than for progesterone as an inhibitor of dehydrogenase activity. Based on these competitive patterns of substrate utilization and product inhibition, the pregnene and androstene activities of 3β-hydroxy-5-ene-steroid dehydrogenase and steroid 5 → 4-ene-isomerase may be expressed at a single catalytic site on one protein in human placenta.