Kinetic evidence for separate 3β-hydroxysteroid dehydrogenase-isomerases in androgen and 16-androstene biosynthetic pathways in the pig testis

Abstract

The microsomal fraction from the testes of immature pigs (<1 week old) contains 3β-hydroxysteroid dehydrogenase-isomerase (3β-HSD-isomerase) activities that convert dehydroepiandrosterone (DHA) to 4-androstenedione and 5,16-androstadien-3β-ol (andien-β) to 4,16-androstadien-3-one (dienone). These reactions are necessary for the biosynthesis of hormonally and pheromonally active steroids. Kinetic analyses of these activities were done to determine whether they are catalysed by a single enzyme or if there is any interaction between the substrates and products of one reaction on the activity of the other enzyme. Kinetic parameters were determined and the affinities for steroid substrate were similar (7–9 μmol/l) but the V max app value for the conversion of andien-β to dienone was 10-fold that of the DHA to 4-androstenedione reaction. In analyses of the conversion of DHA to 4-androstenedione, neither andien-β nor dienone inhibited the reaction and especially, no effect on the K m app for DHA was observed which would have indicated competition between DHA and andien-β for the same active site ( K i app from slope and intercept replots were between 3 and 80 times the values of the kinetic constants). Similarly, DHA and 4-androstenedione had minor or negligible effects on the conversion of andien-β to dienone ( K i app from slope replots were the same as the K m app but the K i app from the intercept replot was 12 to 25% of the V max app ). It is concluded that substrate specific 3β-HSD-isomerases for andien-β and DHA exist in the immature pig testis and there is little, if any interaction between these enzymes.