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]/{Km+[s](1+[s]/Ki)} gave a Km of 0.07±0.01μM, a kcat (for the dimer) of 1.5±0.1s−1, a specificity of 21μM−1·s−1 and a Ki 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 Km, 0.8±0.04s−1 for the kcat 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 (Km = 24±4μM; kcat = 0.47±0.06s−1; specificity = 0.002μM−1·s−1) or DHT (Km = 26±6μM; kcat = 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.

Abbreviations used: 17β-HSD1, human type-1 oestrogenic 17β-hydroxysteroid dehydrogenase; DHEA, dehydroepiandrosterone; DHT, dihydrotestosterone; SDR, short-chain dehydrogenase/reductase.

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