AN ACTIVE-SITE PHENYLALANINE OF 3-OXO-DELTA-(5)-STEROID ISOMERASE IS CATALYTICALLY IMPORTANT FOR PROTON-TRANSFER

3-Oxo-Delta(5)-steroid isomerase (KSI) from Pseudomonas testosteroni catalyzes the isomerization of a variety of 3-oxo-Delta(5)-steroids to their conjugated Delta(4)-isomers through the intermediate formation of a dienolate ion. This dienolate is formed by proton transfer from C-4 of the substrate to Asp-38, which then protonates the dienolate at C-6. Catalysis is enhanced by electrophilic assistance (hydrogen bonding) to the 3-oxygen by Tyr-14. We have investigated the effect of modifying phenylalanine-101 (F101), a hydrophobic residue that is located in the binding pocket of KSI. Two mutant enzymes (F101L and F101A) of KSI were prepared, and their kinetic properties were examined with 5-androstene-3,17-dione (1) as the substrate. Both of the mutants show reduced values of k(cat) compared to the wild type (WT), by about 30-fold (F101L) and by 270-fold (F101A), with only a small difference in K-m values. There is Little change in the K-i's (less than or equal to 4-fold) for the product 4-androstene-3,17-dione (3), although both enzymes bind the intermediate analog d-equilenin (4) about 25-fold less tightly than does the WT. Fluorescence spectra of 4 bound to each of these enzymes suggest that 4 is ionized at the active site of WT, un-ionized at the active site of F101A and a mixture of these ionization states at the active site of F101L. Free energy profiles are constructed for each of the mutant enzymes, and these are compared to the free energy profile for the WT. The results are interpreted in terms of stabilization of the intermediate dienolate and the flanking transition states by the phenyl ring of F101.