This project will continue the study of commonality in the structure and mechanism of steroid-binding proteins. Efforts will focus on three mammalian steroid-interconverting enzymes: 1) human placental estradiol 17beta-dehydrogenase which has been implicated in the physiologic role of maintaining and regulating the appropriate estradiol concentrations in the maternal/fetal unit as delivery approaches; 2) bovine testicular 20alpha-hydroxysteroid dehydrogenase, a multi-functional enzyme identical with aldose reductase which has been suggested to be necessary for sperm maintenance; 3) equine placental estradiol 17alpha/17beta-dehydrogenases which offer the opportunity to investigate the structure/function relationships between two enzymes which utilize epimeric substrates. The studies on the native form of each of these enzymes utilizing affinity labeling steroid substrates (site-directed irreversible inhibitors) will continue with the identification of alkylated residues and the peptides which contain them. In addition, the gene product obtained from cDNA which encodes each enzyme will be examined by affinity labeling techniques and the rigorous kinetic analyses previously applied to the native proteins. Study of point mutations within the cDNA encoding each enzyme will allow specific analysis of structure/function relationships for particular amino acid residues in the altered gene product. There is a continuing, sufficient source available for each native enzyme. The expressed protein for human estradiol 17beta-dehydrogenase and bovine 20alpha-hydroxysteroid dehydrogenase/aldose reductase are currently available as a result of previous efforts in our and collaborating laboratories. Work is presently underway to obtain the cDNA encoding the equine dehydrogenases. The crystalline structure of aldose reductase is now known. The ultimate goal is to validate the affinity labeling the point mutation results with the crystallographic structural studies of the expressed proteins, which will unequivocally define the spatial relationships of amino acid residues, steroid and cofactor at the active site of each of these enzymes.
