Structural and Physiologic Determinants of Estrone/Estradiol Metabolism Catalyzed by Human 17beta -Hydroxysteroid Dehydrogenases Types 1 and 2

dc.contributor.advisorAndersson, Stefanen
dc.creatorSherbet, Daniel P.en
dc.description.abstractThe 17beta -hydroxysteroid dehydrogenases (17beta -HSDs) types 1 and 2 interconvert the weak and potent estrogens estrone and 17beta -estradiol. In intact cells, each enzyme exhibits a strong directional preference that favors either oxidation (17beta -HSD2) or reduction (17beta -HSD1). A positively charged arginine (R38) adjacent to the 2'-phosphate stabilizes NADP(H) binding to 17beta -HSD1 and favors reduction due to the high cytoplasmic NADPH/NADP+ ratio. In contrast, 17beta -HSD2 has a negatively charged glutamate (E116) at the position corresponding to R38 of 17beta -HSD1, which presumably repels the 2'-phosphate of NADP(H) and favors oxidation by harnessing the high cytoplasmic NAD+/NADH ratio. Substitution of a negatively charged aspartate, but not neutral glycine, for R38 of 17beta -HSD1 markedly reduces the affinity for NADP(H) and reverses the directional preference to oxidation in intact cells. We hypothesized that E116 confers oxidative preference to 17beta -HSD2 and that substitution of either a neutral or a positively charged residue for E116 would reverse the directional preference to favor reduction. Mutations E116G, E116R, and the double mutation E116G+N117R failed to attenuate the >95% oxidative preference of 17beta -HSD2 in intact cells. Affinity for all cofactors, as estimated by Km values, were measured for wild-type and mutant 17beta -HSD2 enzymes in yeast microsomes. For wild-type 17beta -HSD2, affinity for NAD(H) is nearly 1000-fold greater than for NADP(H), and the mutant enzymes retain high affinity for NAD(H) yet only slightly better affinity for NADP(H). We conclude that the directional preference of 17beta -HSD1 is principally governed by electrostatic interactions between R38 and the 2'-phosphate of NADP(H), but that the oxidative preference of 17beta -HSD2 is not solely due to E116 in the cofactor-binding domain. These data suggest that the directional preference of 17beta -HSD2 is controlled by other aspects of its cofactor-binding domain, such as the size of the cofactor-binding pocket.en
dc.format.digitalOriginborn digitalen
dc.subjectHydroxysteroid Dehydrogenasesen
dc.subjectMutagenesis, Site-Directeden
dc.titleStructural and Physiologic Determinants of Estrone/Estradiol Metabolism Catalyzed by Human 17beta -Hydroxysteroid Dehydrogenases Types 1 and 2en
dc.type.materialTexten Southwestern Medical Schoolen Southwestern Medical Centeren with Distinctionen


Original bundle

Now showing 1 - 1 of 1
Thumbnail Image
459.28 KB
Adobe Portable Document Format

License bundle

Now showing 1 - 1 of 1
No Thumbnail Available
942 B
Item-specific license agreed upon to submission