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

Human oestrogenic 17 beta -hydroxysteroid dehydrogenase (17 beta- HSD1) cat alyses 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, dehydroepiandrost erone (DHEA) or dihydrotestosterone (DHT) as substrate and NADP(H) as the c ofactor. Kinetic studies made over a wide range of oestrone concentrations (10 nM-10 muM) revealed a typical substrate-inhibition phenomenon. Data ana lysis 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 sit e (Leu36Asp) eliminated the substrate inhibition observed in the presence o f NADPH, confirming the NADPH-dependence of the phenomenon. The possible fo rmation of an enzyme-NADP(+)-oestrone dead-end complex during the substrate -inhibition process is supported by the competitive inhibition of oestradio l 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 specif icities and no substrate inhibition. Taken together, our results demonstrat e that the high specificity of 17 beta -HSD1 towards oestrone is coupled wi th an NADPH-dependent substrate inhibition, suggesting that both the specif icity and the enzyme control are provided for the cognate substrate.