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).[1]References
- Examination of the differences in structure-function of human and rat 3alpha-hydroxysteroid dehydrogenase. Jin, Y., Cooper, W.C., Penning, T.M. Chem. Biol. Interact. (2003) [Pubmed]
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