Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 May 15;356(Pt 1):269–276. doi: 10.1042/0264-6021:3560269

Human oestrogenic 17beta-hydroxysteroid dehydrogenase specificity: enzyme regulation through an NADPH-dependent substrate inhibition towards the highly specific oestrone reduction.

A Gangloff 1, A Garneau 1, Y W Huang 1, F Yang 1, S X Lin 1
PMCID: PMC1221836  PMID: 11336660

Abstract

Human oestrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) catalyses the final step in the biosynthesis of all active oestrogens. Here we report the steady-state kinetics for 17beta-HSD1 at 37 degrees C and pH 7.5, using a homogeneous enzyme preparation with oestrone, dehydroepiandrosterone (DHEA) or dihydrotestosterone (DHT) as substrate and NADP(H) as the cofactor. Kinetic studies made over a wide range of oestrone concentrations (10 nM-10 microM) revealed a typical substrate-inhibition phenomenon. Data analysis using the substrate-inhibition equation v=V.[s]/[K(m)+[s](1+[s]/K(i))] gave a K(m) of 0.07+/-0.01 microM, a k(cat) (for the dimer) of 1.5+/-0.1 s(-1), a specificity of 21 microM(-1) x s(-1) and a K(i) of 1.3 microM. When NADH was used instead of NADPH, substrate inhibition was no longer observed and the kinetic constants were significantly modified to 0.42+/-0.07 microM for the K(m), 0.8+/-0.04 s(-1) for the k(cat) and 1.9 microM(-1) x s(-1) for the specificity. The modification of an amino acid in the cofactor-binding site (Leu36Asp) eliminated the substrate inhibition observed in the presence of NADPH, confirming the NADPH-dependence of the phenomenon. The possible formation of an enzyme-NADP(+)-oestrone dead-end complex during the substrate-inhibition process is supported by the competitive inhibition of oestradiol oxidation by oestrone. Kinetic studies performed with either DHEA (K(m)=24+/-4 microM; k(cat)=0.47+/-0.06 s(-1); specificity=0.002 microM(-1) x s(-1)) or DHT (K(m)=26+/-6 microM; k(cat)=0.2+/-0.02 s(-1); specificity=0.0008 microM(-1) x s(-1)) in the presence of NADP(H) resulted in low specificities and no substrate inhibition. Taken together, our results demonstrate that the high specificity of 17beta-HSD1 towards oestrone is coupled with an NADPH-dependent substrate inhibition, suggesting that both the specificity and the enzyme control are provided for the cognate substrate.

Full Text

The Full Text of this article is available as a PDF (147.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Azzi A., Rehse P. H., Zhu D. W., Campbell R. L., Labrie F., Lin S. X. Crystal structure of human estrogenic 17 beta-hydroxysteroid dehydrogenase complexed with 17 beta-estradiol. Nat Struct Biol. 1996 Aug;3(8):665–668. doi: 10.1038/nsb0896-665. [DOI] [PubMed] [Google Scholar]
  2. Betz G. Reaction mechanism of 17-beta-estradiol dehydrogenase determined by equilibrium rate exchange. J Biol Chem. 1971 Apr 10;246(7):2063–2068. [PubMed] [Google Scholar]
  3. Blomquist C. H., Kotts C. E., Hakanson E. Y. Inhibiton of human placental 17 beta-hydroxysteroid dehydrogenase by steroids and nonsteroidal alcohols: aspects of inhibitor structure and binding specificity. Arch Biochem Biophys. 1978 Feb;186(1):35–41. doi: 10.1016/0003-9861(78)90460-5. [DOI] [PubMed] [Google Scholar]
  4. Bonney R. C., Reed M. J., Beranek P. A., Ghilchik M. W., James V. H. Metabolism of [3H]oestradiol in vivo by normal breast and tumour tissue in postmenopausal women. J Steroid Biochem. 1986 Jan;24(1):361–364. doi: 10.1016/0022-4731(86)90082-8. [DOI] [PubMed] [Google Scholar]
  5. Breton R., Yang F., Jin J. Z., Li B., Labrie F., Lin S. X. Human 17 beta-hydroxysteroid dehydrogenase: overproduction using a baculovirus expression system and characterization. J Steroid Biochem Mol Biol. 1994 Sep;50(5-6):275–282. doi: 10.1016/0960-0760(94)90132-5. [DOI] [PubMed] [Google Scholar]
  6. Duncan L. J., Reed M. J. The role and proposed mechanism by which oestradiol 17 beta-hydroxysteroid dehydrogenase regulates breast tumour oestrogen concentrations. J Steroid Biochem Mol Biol. 1995 Dec;55(5-6):565–572. doi: 10.1016/0960-0760(95)00207-3. [DOI] [PubMed] [Google Scholar]
  7. Eszes C. M., Sessions R. B., Clarke A. R., Moreton K. M., Holbrook J. J. Removal of substrate inhibition in a lactate dehydrogenase from human muscle by a single residue change. FEBS Lett. 1996 Dec 16;399(3):193–197. doi: 10.1016/s0014-5793(96)01317-8. [DOI] [PubMed] [Google Scholar]
  8. Ferre F., Breuiller M., Tanguy G., Janssens Y., Cedard L. Steroid concentrations and delta 5, 3 beta-hydroxysteroid dehydrogenase activity in human placenta. Comparison between elective cesarean section and spontaneous vaginal delivery. Am J Obstet Gynecol. 1980 Nov 1;138(5):500–503. doi: 10.1016/0002-9378(80)90276-8. [DOI] [PubMed] [Google Scholar]
  9. Ghosh D., Pletnev V. Z., Zhu D. W., Wawrzak Z., Duax W. L., Pangborn W., Labrie F., Lin S. X. Structure of human estrogenic 17 beta-hydroxysteroid dehydrogenase at 2.20 A resolution. Structure. 1995 May 15;3(5):503–513. doi: 10.1016/s0969-2126(01)00183-6. [DOI] [PubMed] [Google Scholar]
  10. Greenlee R. T., Murray T., Bolden S., Wingo P. A. Cancer statistics, 2000. CA Cancer J Clin. 2000 Jan-Feb;50(1):7–33. doi: 10.3322/canjclin.50.1.7. [DOI] [PubMed] [Google Scholar]
  11. Han Q., Campbell R. L., Gangloff A., Huang Y. W., Lin S. X. Dehydroepiandrosterone and dihydrotestosterone recognition by human estrogenic 17beta-hydroxysteroid dehydrogenase. C-18/c-19 steroid discrimination and enzyme-induced strain. J Biol Chem. 2000 Jan 14;275(2):1105–1111. doi: 10.1074/jbc.275.2.1105. [DOI] [PubMed] [Google Scholar]
  12. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  14. Jin J. Z., Lin S. X. Human estrogenic 17beta-hydroxysteroid dehydrogenase: predominance of estrone reduction and its induction by NADPH. Biochem Biophys Res Commun. 1999 Jun 7;259(2):489–493. doi: 10.1006/bbrc.1999.0704. [DOI] [PubMed] [Google Scholar]
  15. LANGER L. J., ENGEL L. L. Human placental estradiol-17 beta dehydrogenase. I. Concentration, characterization and assay. J Biol Chem. 1958 Sep;233(3):583–588. [PubMed] [Google Scholar]
  16. Labrie C., Martel C., Dufour J. M., Lévesque C., Mérand Y., Labrie F. Novel compounds inhibit estrogen formation and action. Cancer Res. 1992 Feb 1;52(3):610–615. [PubMed] [Google Scholar]
  17. Lin S. X., Yang F., Jin J. Z., Breton R., Zhu D. W., Luu-The V., Labrie F. Subunit identity of the dimeric 17 beta-hydroxysteroid dehydrogenase from human placenta. J Biol Chem. 1992 Aug 15;267(23):16182–16187. [PubMed] [Google Scholar]
  18. Luu-The V., Zhang Y., Poirier D., Labrie F. Characteristics of human types 1, 2 and 3 17 beta-hydroxysteroid dehydrogenase activities: oxidation/reduction and inhibition. J Steroid Biochem Mol Biol. 1995 Dec;55(5-6):581–587. doi: 10.1016/0960-0760(95)00209-x. [DOI] [PubMed] [Google Scholar]
  19. Ma H., Ratnam K., Penning T. M. Mutation of nicotinamide pocket residues in rat liver 3 alpha-hydroxysteroid dehydrogenase reveals different modes of cofactor binding. Biochemistry. 2000 Jan 11;39(1):102–109. doi: 10.1021/bi991659o. [DOI] [PubMed] [Google Scholar]
  20. Martel C., Rhéaume E., Takahashi M., Trudel C., Couët J., Luu-The V., Simard J., Labrie F. Distribution of 17 beta-hydroxysteroid dehydrogenase gene expression and activity in rat and human tissues. J Steroid Biochem Mol Biol. 1992 Mar;41(3-8):597–603. doi: 10.1016/0960-0760(92)90390-5. [DOI] [PubMed] [Google Scholar]
  21. Mazza C., Breton R., Housset D., Fontecilla-Camps J. C. Unusual charge stabilization of NADP+ in 17beta-hydroxysteroid dehydrogenase. J Biol Chem. 1998 Apr 3;273(14):8145–8152. doi: 10.1074/jbc.273.14.8145. [DOI] [PubMed] [Google Scholar]
  22. Mendoza-Hernández G., Calcagno M., Sánchez-Nuncio H. R., Díaz-Zagoya J. C. Dehydroepiandrosterone is a substrate for estradiol 17 beta-dehydrogenase from human placenta. Biochem Biophys Res Commun. 1984 Feb 29;119(1):83–87. doi: 10.1016/0006-291x(84)91621-8. [DOI] [PubMed] [Google Scholar]
  23. Pocker Y., Raymond K. W. Liver alcohol dehydrogenase: substrate inhibition and competition between substrates. Alcohol. 1985 Jan-Feb;2(1):3–8. doi: 10.1016/0741-8329(85)90004-7. [DOI] [PubMed] [Google Scholar]
  24. Poutanen M., Isomaa V., Lehto V. P., Vihko R. Immunological analysis of 17 beta-hydroxysteroid dehydrogenase in benign and malignant human breast tissue. Int J Cancer. 1992 Feb 1;50(3):386–390. doi: 10.1002/ijc.2910500310. [DOI] [PubMed] [Google Scholar]
  25. Puranen T. J., Poutanen M. H., Peltoketo H. E., Vihko P. T., Vihko R. K. Site-directed mutagenesis of the putative active site of human 17 beta-hydroxysteroid dehydrogenase type 1. Biochem J. 1994 Nov 15;304(Pt 1):289–293. doi: 10.1042/bj3040289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Puranen T., Poutanen M., Ghosh D., Vihko R., Vihko P. Origin of substrate specificity of human and rat 17beta-hydroxysteroid dehydrogenase type 1, using chimeric enzymes and site-directed substitutions. Endocrinology. 1997 Aug;138(8):3532–3539. doi: 10.1210/endo.138.8.5303. [DOI] [PubMed] [Google Scholar]
  27. Roberts P., Basran J., Wilson E. K., Hille R., Scrutton N. S. Redox cycles in trimethylamine dehydrogenase and mechanism of substrate inhibition. Biochemistry. 1999 Nov 9;38(45):14927–14940. doi: 10.1021/bi9914098. [DOI] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Vogel V. G. Breast cancer prevention: a review of current evidence. CA Cancer J Clin. 2000 May-Jun;50(3):156–170. doi: 10.3322/canjclin.50.3.156. [DOI] [PubMed] [Google Scholar]
  30. Zhu D. W., Lee X., Breton R., Ghosh D., Pangborn W., Daux W. L., Lin S. X. Crystallization and preliminary X-ray diffraction analysis of the complex of human placental 17 beta-hydroxysteroid dehydrogenase with NADP+. J Mol Biol. 1993 Nov 5;234(1):242–244. doi: 10.1006/jmbi.1993.1578. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES