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
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
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DOI：
10.1074/jbc.m208537200
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发表时间：
2002-11-08
影响因子：
4.8
通讯作者：
Norris, W
中科院分区：
文献类型：
--
作者：
Thomas, JL;Mason, JI;Norris, W
Two distinct genes encode the 93% homologous type 1 (placenta, peripheral tissues) and type 2 (adrenals, gonads) 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) in humans. Mutagenesis studies using the type 1 enzyme have produced the Y154F and K158Q mutant enzymes in the Y-154-P-H-156-S-K-158 motif as well as the Y269S and K273Q mutants from a second Motif, Y-269-T-L-S-K-273, both of which are present in the primary structure of the human type I 3beta-HSD/isomerase. In addition, the H156Y mutant of the type 1 enzyme has created a chimera of the type 2 enzyme Motif (Y-154-P-Y-156-S-K-158) in the type 1 enzyme. The mutant and wild-type enzymes have been expressed and purified. The K. value of dehydroepiandrosterone is 13-fold greater, and the maximal turnover rate (K-cat) is 2-fold greater for wild-type 2 3beta-HSD compared with the wild-type 1 3beta-HSD activity. The H156Y mutant of the type 1 enzyme has substrate kinetic constants for 3beta-HSD activity that are very similar to those of the wild-type 2 enzyme. Dixon analysis shows that epostane inhibits the 3beta-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 3beta-HSD activity, have substantial isomerase activity, and utilize substrate with K-m values similar to those of wild-type 1 isomerase. The Y269S and K273Q mutants have low, pH-dependent 3beta-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 3beta-HSD, plus a catalytic role for the Tyr(154) and Lys(158) residues in the 3beta-HSD reaction have been identified. These advances in our understanding of the structure/function of human type 1 and 2 3beta-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.
