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J. Biol. Chem., Vol. 282, Issue 46, 33484-33493, November 16, 2007

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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 3-HSD (AKR1C9)^*

William C. Cooper¹, Yi Jin¹, and Trevor M. Penning²

From the Center of Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084

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 3-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 [²H]NADPH, whereas ^Dk_cat/K_m and the ^Dk_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.

Received for publication, April 24, 2007 , and in revised form, September 7, 2007.

^* This work was supported by National Institutes of Health Grants R01-DK47015 and P30 ES013508 (to T. M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Pharmacology, University of Pennsylvania, 130C John Morgan Bldg., 3620 Hamilton Walk, Philadelphia, PA 19104-6084. Tel.: 215-573-2236; Fax: 215-573-2236; E-mail: penning{at}pharm.med.upenn.edu.

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