Delta 5-3-Ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni converts delta 5-3-ketosteroids to delta 4-3-ketosteroids by a stereoselective and conservative transfer of the 4 beta-proton to the 6 beta-position. The 10(9.5)-fold enzymatic rate acceleration can be attributed to a concerted rate-limiting enolization in which Tyr-14 and Asp-38, positioned orthogonally, act as general acid and base, respectively. The pKa value of the phenolic hydroxyl group of Tyr-14 of the Y55F/Y88F double mutant is 11.6 +/- 0.2 by UV titration. However, the fluorescence titration of Tyr-14 shows biphasic sigmoidal behavior with apparent pKa values of 9.5 and 11.5. This suggests the assistance of a basic residue at the active site, possibly a lysine or tyrosine residue. Mutation of each of the four lysine residues K119L, K108Q, K92Q, and K60Q lowered specific activities only slightly to between 43% and 98% of the wild-type enzyme. Similarly, mutations of Tyr-55, Tyr-88, or both to phenylalanine led to only 2-4-fold reductions in catalytic activity. These findings suggest that despite the enormous difference between the pKa value of Tyr-14 (11.6) and that of the 3-carbonyl group of the steroid (ca. pKa-7), the reaction may rely on the concerted participation of Tyr-14 and Asp-38 only. The apparent pKa value of 9.5 in the fluorescence titration of Tyr-14 and in kinetic measurements probably results from conformational changes of the enzyme. The unusually high pKa value of Tyr-14 of 11.6 +/- 0.2 was used to estimate a local dielectric constant of 18 +/- 2 near this residue.(ABSTRACT TRUNCATED AT 250 WORDS)