Differences in substrate and inhibitor kinetics of human type 1 and type 2 3 beta-hydroxysteroid dehydrogenase are explained by the type 1 mutant, H156Y

Two distinct genes encode the human type 1 (placenta, mammary gland) and type 2 (adrenal, gonad) isoforms of 3beta-hydroxysteroid dehydrogenase/ isomerase (3beta-HSD). We have produced the Y154F, H156Y, and K158Q mutant enzymes in the Y-154 -P-H-156-S-K-158 motif of the human type 1 3beta-HSD/ isomerase. The H156Y mutant was created to produce a chimera of the type 2 enzyme motif (Y-154-P-Y-156 -S-K-158) in the type 1 enzyme. The wild-type (WT) 1 and 2 plus the mutant enzymes were expressed and purified. The K-m for dehydroepiandrosterone and K-i for epostane measured with both the H156Y mutant and WT 2 are 13-fold to 17-fold greater than those values obtained with the WT 1 3beta-HSD. The Y154F and K158Q mutants exhibit no 3beta-HSD but have significant isomerase activity. Thus, H-156 in WT 1 vs. Y-156 in WT 2 accounts for the substantially higher affinity of WT 1 3beta-HSD activity for these substrate and inhibitor steroids relative to the WT 2 enzyme.