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 pH7.5, 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 (10nM–10μM) 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±0.01μM, a k_cat (for the dimer) of 1.5±0.1s⁻¹, a specificity of 21μM⁻¹·s⁻¹ and a K_i of 1.3μM. When NADH was used instead of NADPH, substrate inhibition was no longer observed and the kinetic constants were significantly modified to 0.42±0.07μM for the K_m, 0.8±0.04s⁻¹ for the k_cat and 1.9μM⁻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±4μM; k_cat = 0.47±0.06s⁻¹; specificity = 0.002μM⁻¹·s⁻¹) or DHT (K_m = 26±6μM; k_cat = 0.2±0.02s⁻¹; specificity = 0.0008μM⁻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.