This project will continue study of the comparative structure and binding site topography of steroid-binding proteins. We will utilize: (a) affinity-labeling steroids (site directed irreversible inhibitors) and suicide substrates (site-generated irreversible inhibitors) with identification of alkylated residues and peptides that contain them, (b) determination of primary structure (amino acid sequence), so that steroid binding sites (peptides defined in (a)) can be localized and oriented, and (c) X-ray crystallography. For the steroid-interconverting enzymes, we will also: (a) determine stereospecificity of hydride transfer to cofactor, (b) evaluate spatial relationships of steroid and cofactor at the active site, and (c) identify those amino acid residues that proximate the site of the catalytic event and presumably play a role in inducing the transition state. We will study human placental estradiol 17 beta-dehydrogenase, the epimeric estradiol 17 alpha-and 17 beta- dehydrogenases from horse placenta, bovine testicular 20 alpha- hydroxysteroid dehydrogenase (all of which have been purified in this lab and are available in quantities sufficient to carry out these studies) and progesterone binding globulin of guinea pig serum supplied by Dr. Larry Kuhn. No unequivocal evidence exists as to how nature constructs a steroid binding site or the catalytic mechanism by which a steroid interconverting enzyme induces the transition state on its substrate. This project is designed to answer these questions. Our ultimate goal is to carry out X-ray crystallographic studies because we know that this method alone will give unequivocal data as to how active site amino acid residues are spatially related to each other and to the steroid and cofactor at the macromolecular binding site. We will also make wide application of affinity-labeling steroids and suicide substrates, less precise, but more readily applicable methods for elucidating binding site structure. Our basic hypothesis is that, after one has done enough studies with affinity-labeling compounds and suicide substrates with subsequent validation by X-ray crystallogaphy, these more readily applicable techniques may become adequate for at least definition of commonality in steroid binding sites from various sources.