Related Experiment Video
Updated: Jul 11, 2026
Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay
Published on: December 19, 2018
Human oestrogenic 17beta-hydroxysteroid dehydrogenase specificity: enzyme regulation through an NADPH-dependent
A Gangloff1, A Garneau, Y W Huang
1Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL), 2705 Boulevard Laurier, Québec, G1V 4G2, Canada.
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.
More Related Videos
06:18An In Vivo Estrogen Deficiency Mouse Model for Screening Exogenous Estrogen Treatments of Cardiovascular Dysfunction After Menopause
Published on: August 13, 2019
08:00Colorimetric Assessment of Deiodinase 1 Activity in Human Liver Microsomes Using the Sandell-Kolthoff Reaction
Published on: April 10, 2026
Related Concept Videos
01:30Enzyme Inhibition
02:44Oxidation of Alkenes: Syn Dihydroxylation with Osmium Tetraoxide
02:17Reduction of Alkenes: Asymmetric Catalytic Hydrogenation
The metal catalyst used can be either heterogeneous or homogeneous. When hydrogenation of an alkene generates a chiral center, a pair of enantiomeric products is expected to form. However, an enantiomeric excess of one of the products can be facilitated using an enantioselective reaction or an...
01:31Transducer Mechanism: Nuclear Receptors
About 48 different soluble family members of nuclear receptors are identified that can be divided into two main classes:
01:22Hormonal Regulation of the Menstrual Cycle
At puberty, GnRH begins a pulsatile release pattern, which triggers the anterior pituitary gland to secrete follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The frequency and amplitude of GnRH pulses vary across the menstrual cycle, with faster pulses favoring LH release and slower pulses favoring FSH release.
01:30Hormonal Control of the Ovarian Cycle
Before puberty, the hypothalamus releases GnRH in a low frequency, low amplitude pulsatile manner. This along with the immature hypothalamic-pituitary-gonadal axis activity, results in low estrogen levels and the absence of a fully functional ovarian cycle. At puberty, GnRH secretion increases in both frequency and...