Δ5-3-Ketosteroid isomerase (EC 18.104.22.168) from Pseudomonas testosteroni converts Δ5-3-ketosteroids to Δ4-3-ketosteroids by a stereoselective and conservative transfer of the 4β-proton to the 6β-position. The 109.5-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. Mutations 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. UV absorption spectra of each of the three tyrosine double and single mutants reveal that the molar absorbances of Tyr-14 and Tyr-55 are 25–30% greater than those of Tyr-88 or of N-acetyltyrosine amide in solution and are red-shifted by 4–5 nm. Fluorescence studies show that the relative molar fluorescence intensity of Tyr-14 is unusually high (3.9) and that of Tyr-88 is much lower (0.42) in comparison to that of free N-acetyltyrosine amide. Consistent with the estimated dielectric constant of 18 ± 2 near Tyr-14, both the UV absorption spectrum and the fluorescence enhancement of Tyr-14 are mimicked by N-acetyltyrosine amide in 2-propanol, a solvent with a dielectric constant of 18.6. The fluorescences of Tyr-14 and Tyr-55 are strongly mutually quenched, whereas Tyr-88 has no effect on the fluorescence of these residues, and vice versa. The binding of 19-nortestosterone to the enzyme quenches nearly all of the fluorescence of Tyr-14 and Tyr-55, but only partially quenches the fluorescence of Tyr-88. These findings are consistent with the partial X-ray structure of isomerase which reveals that Tyr-14 and Tyr-55 are located in the hydrophobic active site while Tyr-88 is more distant and at least partially exposed to the medium.