Abstract

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