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.

Alanine scanning mutagenesis of the testosterone binding site of rat 3 alpha-hydroxysteroid dehydrogenase demonstrates contact residues influence the rate-determining step.

Aldo-keto reductase (AKR1C) isoforms can regulate ligand access to nuclear receptors by acting as hydroxysteroid dehydrogenases. The principles that govern steroid hormone binding and steroid turnover by these enzymes were analyzed using rat 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD, AKR1C9) as the protein model. Systematic alanine scanning mutagenesis was performed on the substrate-binding pocket as defined by the crystal structure of the 3alpha-HSD.NADP(+).testosterone ternary complex. T24, L54, F118, F129, T226, W227, N306, and Y310 were individually mutated to alanine, while catalytic residues Y55 and H117 were unaltered. The effects of these mutations on the ordered bi-bi mechanism were examined. No mutations changed the affinity for NADPH by more than 2-3-fold. Fluorescence titrations of the energy transfer band of the E.NADPH complex with competitive inhibitors testosterone and progesterone showed that the largest effect was a 23-fold decrease in the affinity for progesterone in the W227A mutant. By contrast, changes in the K(d) for testosterone were negligible. Examination of the k(cat)/K(m) data for these mutants indicated that, irrespective of steroid substrate, the bimolecular rate constant was more adversely affected when alanine replaced an aromatic hydrophobic residue. By far, the greatest effects were on k(cat) (decreases of more than 2 log units), suggesting that the rate-determining step was either altered or slowed significantly. Single- and multiple-turnover experiments for androsterone oxidation showed that while the wild-type enzyme demonstrated a k(lim) and burst kinetics consistent with slow product release, the W227A and F118A mutants eliminated this kinetic profile. Instead, single- and multiple-turnover experiments gave k(lim) and k(max) values identical with k(cat) values, respectively, indicating that chemistry was now rate-limiting overall. Thus, conserved residues within the steroid-binding pocket affect k(cat) more than K(d) by influencing the rate-determining step of steroid oxidation. These findings support the concept of enzyme catalysis in which the correct positioning of reactants is essential; otherwise, k(cat) will be limited by the chemical event.

Examination of the differences in structure-function of human and rat 3alpha-hydroxysteroid dehydrogenase.

Human 3alpha-hydroxysteroid dehydrogenases (HSDs) are potential drug targets since they regulate the occupancy and trans-activation of steroid hormone receptors by interconverting potent hormones with their cognate inactive metabolites. The human isoforms (AKR1C1-4) are all members of the aldo-keto reductase superfamily and display distinctive differences in steroid specificity and catalytic efficiency when compared with the closely related and more extensively studied rat 3alpha-HSD (AKR1C9). Specifically, AKR1C1-4 display 3alpha-, 17beta- and 20alpha-HSD activities to varying degrees whereas AKR1C9 is positional- and stereo-specific for the 3alpha-HSD reaction. In addition, AKR1C1-4 isoforms have significantly lower catalytic efficiencies (k(cat)/K(m)) than AKR1C9 and this is largely due to a lower k(cat). To understand these functional differences, human type 3 3alpha-HSD (AKR1C2) was studied as a representative human 3alpha-HSD. Comparison of the crystal structure of AKR1C2-NADP(+)-ursodeoxycholate ternary complex (3.0 A) with that of the AKR1C9-NADP(+)-testosterone ternary complex (2.8 A) demonstrates the expected conservancy in overall structure and active site topology. More interestingly, it reveals striking differences in the structure of the steroid binding pockets of the two enzymes and shows how ursodeoxycholate binds 'backwards' and 'upside-down' with respect to testosterone. This difference in steroid binding provides a structural basis for the broad positional specificity of AKR1C2 and the exquisite stereospecificity of AKR1C9. To determine why AKR1C2 has a much lower k(cat) than AKR1C9, the events associated with the binding of cofactor to both enzymes were studied by steady state fluorescence titration and stopped-flow experiments. Comparable K(d) values for E-NADP(H) and k(obs) values for the fluorescence transients were obtained for the two enzymes. These data are consistent with both enzymes binding NADP(H) in a conserved manner which is supported by the available crystal structures. The results suggest that cofactor binding or release for the human and rat 3alpha-HSDs are similar and do not account for the observed differences in k(cat).