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Δ^(5)-3-Ketosteroid isomerase (KSI) promotes the highly efficient isomerization of Δ^(5)-3-ketosteroids to Δ⁴-3-ketosteroids by means of a direct and stereospecific transfer of the 4β-proton to the 6β-position. In order to compare the catalytic mechanisms of Pseudomonas putida isomerase with Comamonas testosteroni isomerase, well-conserved putative active-site amino acids in the P. putida KSI were replaced by site-directed mutagenesis: Asp-40 to asparagine and Tyr-16 to phenylalanine (D40N + Y16F). The double mutant isomerase was purified to homogeneity and characterized. The molecular weight of the purified double mutant was 14,517 (calculated, 14,519) as determined by electrospray mass spectrometry. While C. testosteroni D38N + Y14F double mutant showed no catalytic activity [Kuliopulos, A., Talalay, P., and Mildvan, A. S. (1990) Biochemistry 29, 10271-10280], P. putida D40N + Y16F double mutant had some detectable activity. The k_(cat) value of the double mutant enzyme is 10^(6.6)-fold lower than that of the wild-type enzyme and K_(m) value is similar to that of wild-type enzyme. Although active-site residues of the two homologous enzymes are well conserved and of similar properties, kinetic properties of P. putida double mutant isomerase are not similar to those of the C. testosteroni double mutant. This result suggests that the precise mechanism of both homologous enzymes are somewhat different.
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