Human oestrogenic 17beta-hydroxysteroid dehydrogenase specificity: enzyme regulation through an NADPH-dependent substrate inhibition towards the highly specific oestrone reduction.
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.
Gangloff
A
A
Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL), 2705 Boulevard Laurier, Québec, G1V 4G2, Canada.
Garneau
A
A
Huang
Y W
YW
Yang
F
F
Lin
S X
SX
D015067:17-Hydroxysteroid Dehydrogenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000235:genetics; D001798:Blood Proteins; D003687:Dehydroepiandrosterone / Q000378:metabolism; D013196:Dihydrotestosterone / Q000378:metabolism; D004967:Estrogens / Q000096:biosynthesis*; D004970:Estrone / Q000378:metabolism*; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D007930:Leucine / Q000235:genetics; D008565:Membrane Proteins; D008956:Models, Chemical; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy
D001798:Blood Proteins; D004967:Estrogens; D007527:Isoenzymes; C088547:KTN1 protein, human; D008565:Membrane Proteins; D013196:Dihydrotestosterone; D009243:NAD; D004970:Estrone; D001224:Aspartic Acid; D003687:Dehydroepiandrosterone; D009249:NADP; D015067:17-Hydroxysteroid Dehydrogenases; C044256:3 (or 17)-beta-hydroxysteroid dehydrogenase; D007930:Leucine