Elucidation of a complete kinetic mechanism for a mammalian hydroxysteroid dehydrogenase (HSD) and identification of all enzyme forms on the reaction coordinate: the example of rat liver 3alpha-HSD (AKR1C9).
Hydroxysteroid dehydrogenases (HSDs) are essential for the biosynthesis and mechanism of action of all steroid hormones. We report the complete kinetic mechanism of a mammalian HSD using rat 3alpha-HSD of the aldo-keto reductase superfamily (AKR1C9) with the substrate pairs androstane-3,17-dione and NADPH (reduction) and androsterone and NADP(+) (oxidation). Steady-state, transient state kinetics, and kinetic isotope effects reconciled the ordered bi-bi mechanism, which contained 9 enzyme forms and permitted the estimation of 16 kinetic constants. In both reactions, loose association of the NADP(H) was followed by two conformational changes, which increased cofactor affinity by >86-fold. For androstane-3,17-dione reduction, the release of NADP(+) controlled k(cat), whereas the chemical event also contributed to this term. k(cat) was insensitive to [(2)H]NADPH, whereas (D)k(cat)/K(m) and the (D)k(lim) (ratio of the maximum rates of single turnover) were 1.06 and 2.06, respectively. Under multiple turnover conditions partial burst kinetics were observed. For androsterone oxidation, the rate of NADPH release dominated k(cat), whereas the rates of the chemical event and the release of androstane-3,17-dione were 50-fold greater. Under multiple turnover conditions full burst kinetics were observed. Although the internal equilibrium constant favored oxidation, the overall K(eq) favored reduction. The kinetic Haldane and free energy diagram confirmed that K(eq) was governed by ligand binding terms that favored the reduction reactants. Thus, HSDs in the aldo-keto reductase superfamily thermodynamically favor ketosteroid reduction.
Cooper
William C
WC
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084.
Jin
Yi
Y
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084.
Penning
Trevor M
TM
Center of Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, 19104-6084. Electronic address: penning@pharm.med.upenn.edu.
D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific) / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D018360:Crystallography, X-Ray; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D009249:NADP / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D011487:Protein Conformation; D051381:Rats; D013739:Testosterone / Q000494:pharmacology; D013816:Thermodynamics; D013997:Time Factors
D013739:Testosterone; D009249:NADP; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific); D010100:Oxygen