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

Abstract

Human oestrogenic 17β-hydroxysteroid dehydrogenase (17β-HSD1) catalyses the final step in the biosynthesis of all active oestrogens. Here we report the steady-state kinetics for 17β-HSD1 at 37°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 μM) revealed a typical substrate-inhibition phenomenon. Data analysis using the substrate-inhibition equation v = V·[s]/{Km+[s](1+[s]/K)} gave a Km of 0.07 ± 0.01 μM, a kcat (for the dimer) of 1.5 ± 0.1 s-1, a specificity of 21 μM-1·s-1 and a K1 of 1.3 μM. 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 μM for the Km, 0.8 ± 0.04 s-1 for the kcat and 1.9 μM-1·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 enzym-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 (Km = 24 ± 4 μM; kcat = 0.47 ± 0.06 s-1; specificity = 0.002 μM-1·s-1) or DHT (Km = 26 ± 6 μM; kcat = 0.2 ± 0.02 s-1; specificity = 0.0008 μM-1·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 17β-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.