Catalytic contribution of phenylalanine-101 of 3-oxo-Delta 5-steroid isomerase

3-Oxo-delta 5-steroid isomerase (KSI, EC 5.3.3.1) from Pseudomonas testosteroni catalyzes the isomerization of a variety of 3-oxo-delta 5-steroids to their conjugated Delta4-isomers through the formation of an intermediate dienolate ion. It has previously been found in our laboratory that the aromatic ring of Phe-101 is important for catalysis. The present work extends these studies. Two double-mutant KSIs (D38E/F101L and D38E/F101A) were prepared to compare the free energy profiles for the reactions catalyzed by these mutants and by D38E. Both double-mutant KSIs show reduced values of kcat at pH 7 compared to D38E ( approximately 25-fold for D38E/F101L and approximately 200-fold for D38E/F101A), similar to the reduced values for F101L and F101A relative to KSI ( approximately 30-fold for F101L and approximately 270-fold for F101A). Free energy profiles for the reactions catalyzed by D38E/F101L and D38E/F101A indicate that the bound transition state(s) and bound intermediate are destabilized when the large aromatic residue Phe-101 in D38E KSI is replaced by the smaller residues Leu or Ala. The pH-rate profiles for D38E, D38E/F101L, and D38E/F101A in the pH range 3.9-8.7 show that the pKa of the catalytic base (Glu-38) is perturbed. In addition, these mutants have significant catalytic activity in the low-pH region.