Dissection of the physiological interconversion of 5alpha-DHT and 3alpha-diol by rat 3alpha-HSD via transient kinetics shows that the chemical step is rate-determining: effect of mutating cofactor and substrate-binding pocket residues on catalysis

Biochemistry
Vladi V Heredia, Trevor M Penning

Abstract

3Alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) catalyze the interconversion between 5alpha-dihydrotestosterone (5alpha-DHT), the most potent androgen, and 3alpha-androstanediol (3alpha-diol), a weak androgen metabolite. To identify the rate-determining step in this physiologically important reaction, rat liver 3alpha-HSD (AKR1C9) was used as the protein model for the human homologues in fluorescence stopped-flow transient kinetic and kinetic isotope effect studies. Using single and multiple turnover experiments to monitor the NADPH-dependent reduction of 5alpha-DHT, it was found that k(lim) and k(max) values were identical to k(cat), indicating that chemistry is rate-limiting overall. Kinetic isotope effect measurements, which gave (D)k(cat) = 2.4 and (D)2(O)k(cat) = 3.0 at pL 6.0, suggest that the slow chemical transformation is significantly rate-limiting. When the NADP(+)-dependent oxidation of 3alpha-diol was monitored, single and multiple turnover experiments showed a k(lim) and burst kinetics consistent with product release as being rate-limiting overall. When NAD(+) was substituted for NADP(+), burst phase kinetics was eliminated, and k(max) was identical to k(cat). Thus with the physiologically relevant substrates 5...Continue Reading

Citations

Mar 30, 2006·The Journal of Biological Chemistry·Jun WengMing Zhou
Sep 15, 2006·Annual Review of Pharmacology and Toxicology·Yi Jin, Trevor M Penning
Oct 25, 2008·Drug Metabolism Reviews·Oleg A BarskiAruni Bhatnagar
Jan 16, 2007·Molecular and Cellular Endocrinology·Daniel P SherbetRichard J Auchus
Oct 1, 2008·Molecular and Cellular Endocrinology·Dario Mizrachi, Richard J Auchus
Nov 26, 2015·The Journal of Steroid Biochemistry and Molecular Biology·Trevor M Penning
Jun 1, 2007·Archives of Biochemistry and Biophysics·Trevor M Penning, Jason E Drury

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