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

Human oestrogenic 17 beta -hydroxysteroid dehydrogenase (17 beta- HSD1) catalyses the final step in the biosynthesis of all active oestrogens. Here we report the steady-state kinetics for 17 beta -HSD1 at 37 degreesC 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 muM) 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 muM, a k(cat) (for the dimer) of 1.5 +/- 0.1 s(-1), a specificity of 21 muM(-1).s(-1) and a K-i of 1.3 muM. 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 muM for the K-m, 0.8 +/- 0.04 s(-1) for the k(cat) and 1.9 muM(-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 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 muM; k(cat) = 0.47 +/- 0.06 s(-1); specificity = 0.002 muM(-1) . s(-1)) or DHT (K-m = 26 +/- 6 muM; k(cat) = 0.2 +/- 0.02 s(-1); specificity = 0.0008 muM(-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 17 beta -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.