3-BETA-HYDROXYSTEROID DEHYDROGENASE-ACTIVITY IN TISSUES OF THE HUMAN FETUS DETERMINED WITH 5-ALPHA-ANDROSTANE-3-BETA, 17-BETA-DIOL AND DEHYDROEPIANDROSTERONE AS SUBSTRATES

3beta-Hydroxysteroid dehydrogenase (3beta-HSD)/DELTA5-->4-isomerase activity in steroidogenic tissues is required for the synthesis of biologically active steroids. Previously, by use of dehydroepiandrosterone (3beta-hydroxy-5-androsten-17-one, DHEA) as substrate, it was established that in addition to steroidogenic tissues 3beta-HSD/DELTA5-->4-isomerase activity also is expressed in extraglandular tissues of the human fetus. In the present study, we attempted to determine whether the C-5,C-6-double bond of DHEA serves to influence 3beta-HSD activity. For this purpose, we compared the efficiencies of a 3beta-hydroxy-5-ene steroid (DHEA) and a 3beta-hydroxy-5alpha-reduced steroid (5alpha-androstane-3beta,17beta-diol, 5alpha-A-diol) as substrates for the enzyme. The apparent Michaelis constant (K(m)) for 5alpha-A-diol in midtrimester placenta, fetal liver, and fetal skin tissues was at least one order of magnitude higher than that for DHEA, viz the apparent K(m) of placental 3beta-HSD for 5alpha-A-diol was in the range of 18 to 40 mumol/l (n = 3) vs 0.45 to 4 mumol/l for DHEA (n = 3); for the liver enzyme, 17 mumol/l for 5alpha-A-diol and 0.60 mumol/l for DHEA, and for the skin enzyme 14 and 0.18 mumol/l, respectively. Moreover, in 13 human fetal tissues evaluated the maximal velocities obtained with 5alpha-A-diol as substrate were higher than those obtained with DHEA. A similar finding in regard to K(m)s and rates of product formation was obtained by use of purified placental 3beta-HSD with DHEA, pregnenolone, and 3beta-hydroxy-5alpha-androstan-17-one (epiandrosterone) as substrates: the K. of 3beta-HSD for DHEA was 2.8 mumol/l, for pregnenolone 1.9 mumol/l, and for epiandrosterone 25 mumol/l. The specific activity of the purified enzyme with pregnenolone as substrate was 27 nmol/mg protein - min and, with epiandrosterone, 127 nmol/mg protein - min. With placental homogenate as the source of 3beta-HSD, DHEA at a constant level of 5 mumol/l behaved as a competitive inhibitor when the radiolabeled substrate, [H-3]5alpha-A-diol, was present in concentrations of 20 to 60 mumol/l, but at lower substrate concentrations the inhibition was of the mixed type; similar results were obtained with [H-3]DHEA as the substrate at variable concentrations in the presence of a fixed concentration of 5alpha-A-diol (40 mumol/l). These findings are indicative that both steroids bind to a common site on the enzyme, however, the binding affinity for these steroids appear to differ markedly as suggested by the respective K(m)s. Studies of inactivation of purified placental 3beta-HSD/DELTA5-->4-isomerase by an irreversible inhibitor, viz 5,10-secoestr-4-yne-3,10,17-trione, were suggestive that the placental protein adopts different conformations depending on whether the steroidal substrate has a 5alpha-configuration, e.g. epiandrosterone, or a C-5,C-6-double bond, e.g. DHEA or pregnenolone. The lower rates of product formation obtained with placenta and fetal tissues by use of 3beta-hydroxy-5-ene steroids as substrates when compared with those obtained with 3beta-hydroxy-5alpha-reduced steroids may be explained by a combination of factors, including: (i) inhibition of 3beta-HSD activity by end products of metabolism of 3beta-hydroxy-5-ene steroids, e.g. 4-androstene-3,17-dione formed with DHEA as substrate; (ii) higher binding affinity of the enzyme for 3beta-hydroxy-5-ene steroids-and possibly for their 3-oxo-5-ene metabolites; (iii) lack of a requirement for the isomerization step with 5alpha-reduced steroids as substrates, and (iv) the possible presence in fetal tissues of an enzyme with 3beta-HSD activity only (i.e. no DELTA5-->4-isomerase).