Human 17β-hydroxysteroid dehydrogenases types 1, 2, and 3 catalyze bi-directional equilibrium reactions, rather than unidirectional metabolism, in HEK-293 cells

Abstract

Human 17β-hydroxysteroid dehydrogenases (17βHSDs) 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 17βHSD1 and 17β HSD3 and only oxidative reactions for 17βHSD2. We show that, after exhaustive incubations with either 17β-hydroxy- or 17-ketosteroid, the medium for HEK-293 cells expressing 17βHSD1 or 17βHSD3 contains a 92:8 ratio of reduced:oxidized steroid. Similarly, 17βHSD2 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 17βHSD isoforms, and these rates are much faster than those estimated from single-isotope flux studies. Mutation L36D converts 17βHSD1 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 17βHSD2 paradoxically increases the potency of estrone in transactivation assays, demonstrating the physiological relevance of “backwards” metabolism to estradiol. We conclude that 17βHSD 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 17βHSD isoforms do not drive steroid flux in one direction but rather may achieve functional equilibria in intact cells, reflecting thermodynamically driven steroid distributions.