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 c atalyzes the isomerization of a variety of 3-oxo-Delta(5)-steroids to their conjugated Delta(4)-isomers through the intermediate formation o f 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 bondin g) to the 3-oxygen by Tyr-14. We have investigated the effect of modif ying phenylalanine-101 (F101), a hydrophobic residue that is located i n the binding pocket of KSI. Two mutant enzymes (F101L and F101A) of K SI were prepared, and their kinetic properties were examined with 5-an drostene-3,17-dione (1) as the substrate. Both of the mutants show red uced 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-f old) for the product 4-androstene-3,17-dione (3), although both enzyme s bind the intermediate analog d-equilenin (4) about 25-fold less tigh tly 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-ionize d 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 stabiliza tion of the intermediate dienolate and the flanking transition states by the phenyl ring of F101.