Structure/function relationships responsible for the kinetic differences between human type 1 and type 2 3β-hydroxysteroid dehydrogenase and for the catalysis of the type 1 activity

Abstract

Two distinct genes encode the 93% homologous type 1 (placenta, peripheral tissues) and type 2 (adrenals, gonads) 3β-hydroxysteroid dehydrogenase/isomerase (3β-HSD/isomerase) in humans. Mutagenesis studies using the type 1 enzyme have produced the Y154F and K158Q mutant enzymes in the Y154-P-H156-S-K158 motif as well as the Y269S and K273Q mutants from a second motif, Y269-T-L-S-K273, both of which are present in the primary structure of the human type 1 3β-HSD/isomerase. In addition, the H156Y mutant of the type 1 enzyme has created a chimera of the type 2 enzyme motif (Y154-P-Y156-S-K158) in the type 1 enzyme. The mutant and wild-type enzymes have been expressed and purified. The Km value of dehydroepiandrosterone is 13-fold greater, and the maximal turnover rate (Kcat) is 2-fold greater for wild-type 2 3β-HSD compared with the wild-type 1 3β-HSD activity. The H156Y mutant of the type 1 enzyme has substrate kinetic constants for 3β-HSD activity that are very similar to those of the wild-type 2 enzyme. Dixon analysis shows that epostane inhibits the 3β-HSD activity of the wildtype 1 enzyme with 14-17-fold greater affinity compared with the wild-type 2 and H156Y enzymes. The Y154F and K158Q mutants exhibit no 3β-HSD activity, have substantial isomerase activity, and utilize substrate with Km values similar to those of wild-type 1 isomerase. The Y269S and K273Q mutants have low, pH-dependent 3β-HSD activity, exhibit only 5% of the maximal isomerase activity, and utilize the isomerase substrate very poorly. From these studies, a structural basis for the profound differences in the substrate and inhibition kinetics of the wildtype 1 and 2 3β-HSD, plus a catalytic role for the Tyr154 and Lys158 residues in the 3β-HSD reaction have been identified. These advances in our understanding of the structure/function of human type 1 and 2 3β-HSD/isomerase may lead to the design of selective inhibitors of the type 1 enzyme not only in placenta to control the onset of labor but also in hormone-sensitive breast, prostate, and choriocarcinoma tumors to slow their growth.