Identification of α-Ketobutyrate as the Prosthetic Group of Urocanase from Pseudomonas putida

Urocanase was purified 60-fold and crystallized from extracts of Pseudomonas putida. Crystalline preparations of the enzyme were homogeneous by ultracentrifugal and electrophoretic criteria. Urocanase has an s20,w of 7.6 S, and a molecular weight of 110,000 ± 4,000 was calculated from sedimentation equilibrium data. Electrophoresis of the enzyme in 4 m urea resulted in a dissociation of the native protein into two electrophoretically distinct fractions. Urocanase has a specific activity of 5.1 µmoles of urocanate degraded per min per mg of protein when measured at pH 7.5 and extrapolated to saturating substrate concentration. The Michaelis constant is 0.24 mm. Urocanase was inactivated upon treatment with carbonyl inhibitors such as hydroxylamine and semicarbazide, which suggests that an enzyme-bound carbonyl group was required for the catalytic process. Pyridoxal phosphate was ruled out as a cofactor on the basis of enzyme spectral measurements and direct pyridoxal phosphate analysis. Reduction of urocanase with tritiated sodium borohydride resulted in the complete inactivation of the enzyme and led to the stable incorporation of slightly less than 1 g atom of hydrogen per mole of enzyme. Hydrolysis of labeled enzyme released a radioactive compound which was identified as α-hydroxybutyrate by paper chromatography, by high voltage electrophoresis, and by formation of a p-bromophenacyl ester identical with one prepared from authentic α-hydroxybutyrate. These results indicate that the native urocanase contains a covalently bound α-ketobutyrate which is essential for catalytic activity. A mechanism for urocanase action is presented to explain the role of α-ketobutyrate in the reaction process.