title: Biochemical factors governing the steady-state estrone/estradiol ratios catalyzed by human 17beta-hydroxysteroid dehydrogenases types 1 and 2 in HEK-293 cells.
abstract: Human 17beta-hydroxysteroid dehydrogenase types 1 and 2 (17betaHSD1 and 17betaHSD2) regulate estrogen potency by catalyzing the interconversion of estrone (E1) and estradiol (E2) using nicotinamide adenine dinucleotide (phosphate) cofactors NAD(P)(H). In intact cells, 17betaHSD1 and 17betaHSD2 establish pseudo-equilibria favoring E1 reduction or E2 oxidation, respectively. The vulnerability of these equilibrium steroid distributions to mutations and to altered intracellular cofactor abundance and redox state, however, is not known. We demonstrate that the equilibrium E2/E1 ratio achieved by 17betaHSD1 in intact HEK-293 cell lines is progressively reduced from 94:6 to 10:90 after mutagenesis of R38, which interacts with the 2'-phosphate of NADP(H), and by glucose deprivation, which lowers the NADPH/NADP(+) ratio. The shift to E2 oxidation parallels changes in apparent K(m) values for purified 17betaHSD1 proteins to favor NAD(H) over NADP(H). In contrast, mutagenesis of E116 (corresponding to R38 in 17betaHSD1) and changes in intracellular cofactor ratios do not alter the greater than 90:10 E1/E2 ratio catalyzed by 17betaHSD2, and these mutations lower the apparent K(m) of recombinant 17betaHSD2 for NADP(H) only less than 3-fold. We conclude that the equilibrium E1/E2 ratio maintained by human 17betaHSD1 in intact cells is governed by NADPH saturation, which is strongly dependent on both R38 and high intracellular NADPH/NADP(+) ratios. In contrast, the preference of 17betaHSD2 for E2 oxidation strongly resists alteration by genetic and metabolic manipulations. These findings suggest that additional structural features, beyond the lack of a specific arginine residue, disfavor NADPH binding and thus support E2 oxidation by 17betaHSD2 in intact cells.
authors: Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8857, USA.
authors: Daniel P
authors: DP
authors: Sherbet
authors: Oleg L
authors: OL
authors: Guryev
authors: Mahboubeh
authors: M
authors: Papari-Zareei
authors: Dario
authors: D
authors: Mizrachi
authors: Siayareh
authors: S
authors: Rambally
authors: Sharareh
authors: S
authors: Akbar
authors: Richard J
authors: RJ
authors: Auchus
mesh_terms: D015067:17-Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D003001:Cloning, Molecular; D004958:Estradiol / Q000378:metabolism; D004960:Estradiol Dehydrogenases / Q000378:metabolism*; D004970:Estrone / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D004970:Estrone; D004958:Estradiol; D009249:NADP; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase; D004960:Estradiol Dehydrogenases