DIFFERENTIAL INHIBITION OF DEHYDROGENASE AND 5-ENE-]4-ENE ISOMERASE ACTIVITIES OF PURIFIED 3-BETA-HYDROXYSTEROID DEHYDROGENASE - EVIDENCE FOR 2 DISTINCT SITES

The success in synthesis of [H-3]5-androstene-3,17-dione, the intermediate product in the transformation of DHEA to 4-androstenedione by 3-beta-hydroxysteroid dehydrogenase/5-ene --> 4-ene isomerase (3-beta-HSD) offers the opportunity to determine whether or not the two activities reside in one active site or in two closely related active sites. The finding that N,N-dimethyl-4-methyl-3-oxo-4-aza-5-alpha-androstane-17-beta-carboxamide (4-MA) inhibits competitively and specifically the dehydrogenase activity whereas a non-competitive inhibition type with a K(i) value 1000 fold higher was observed for the isomerase activity, indicated that dehydrogenase and isomerase activities belong to separate sites. Using 5-alpha-dihydro-testosterone and 5-alpha-androstane-3-beta, 17-beta-diol, exclusive substrates for dehydrogenase activity, it was shown that dehydrogenase is reversible and strongly inhibited by 4-MA and that thus the irreversible step in the transformation of DHEA to 4-androstenedione is due to the isomerase activity.