This research proposal's long-term objective is to determine the structural basis for the mechanism of catalysis utilized by the delta-5-3-ketosteroid isomerases from two bacterial species, Pseudomonas testosteroni and Pseudomonas putida. Using electrophilic and photochemical affinity reagents both in free solution and as solid phase conjugates with agarose beads, residues proximal to the A/B rings of bound steroid substrates will be identified. Particular attention will be paid to 10 beta reagent derivatives which are designed to react with the putative proton carrying base thought to be involved in the isomerization reaction catalyzed by these enzymes. Residues presently believed to be at the active site as well as additional residues identified by the proposed affinity labeling studies will be subjected to modification using oligonucleotide- directed mutagenesis of the isomerase gene. Thus, the isomerase gene will be cloned and its nucleotide sequence determined by the dideoxy method applied to M13 recombinants. The mutant genes will be expressed in a suitable host and the mutant proteins purified and their functional properties determined. The identity of aromatic amino acid residues whose aromatic protons are perturbed by steroid binding, as seen by H-NMR spectroscopy, will be identified using similar mutagenesis methodology in which individual aromatic residues are replaced by similar aliphatic residues and a comparison of native and mutant isomerase NMR spectra made.
