Human 17beta-hydroxysteroid dehydrogenases types 1, 2, and 3 catalyze bi-directional equilibrium reactions, rather than unidirectional metabolism, in HEK-293 cells.
Human 17beta-hydroxysteroid dehydrogenases (17betaHSDs) catalyze the interconversion of weak and potent androgen and estrogen pairs. Although the reactions using purified enzymes can be driven in either direction, these enzymes appear to function unidirectionally in intact cells: only reductive reactions for 17betaHSD1 and 17beta HSD3 and only oxidative reactions for 17betaHSD2. We show that, after exhaustive incubations with either 17beta-hydroxy- or 17-ketosteroid, the medium for HEK-293 cells expressing 17betaHSD1 or 17betaHSD3 contains a 92:8 ratio of reduced:oxidized steroid. Similarly, 17betaHSD2 yields a >95:5 ratio of oxidized:reduced steroids for both androgens and estrogens. Dual-isotope kinetic measurements show that the rates of the forward and reverse reactions are identical at these functional equilibrium states in intact cells for all three 17betaHSD isoforms, and these rates are much faster than those estimated from single-isotope flux studies. Mutation L36D converts 17betaHSD1 to an oxidative enzyme in intact cells, reversing the equilibrium distribution of estradiol:estrone to 5:95; however, the rates of the forward and reverse reactions at equilibrium are equal and comparable to those of the wild-type enzymes. The co-expression of 17betaHSD2 paradoxically increases the potency of estrone in transactivation assays, demonstrating the physiological relevance of "backwards" metabolism to estradiol. We conclude that 17betaHSD types 1, 2, and 3 catalyze both oxidative and reductive reactions in HEK-293 cells at intrinsic rates that are much faster than those estimated from single-isotope studies. These 17betaHSD isoforms do not drive steroid flux in one direction but rather may achieve functional equilibria in intact cells, reflecting thermodynamically driven steroid distributions.
Khan
Naveed
N
Divison of Reproductive Sciences, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8857, USA.
Sharma
Kamalesh K
KK
Andersson
Stefan
S
Auchus
Richard J
RJ
D015067:17-Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000201:enzymology*; D007700:Kinetics; D008657:Metabolic Clearance Rate; D011994:Recombinant Proteins / Q000378:metabolism; D013256:Steroids / Q000378:metabolism*; D013379:Substrate Specificity; D014162:Transfection
D011994:Recombinant Proteins; D013256:Steroids; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase