Specific inhibition of bacterial and bovine urocanases by glycylglycine

Urocanase (EC 4.2.1.49) purified from Pseudomonas putida was unexpectedly inhibited by the dipeptide glycylglycine. Using a spectrophotometric assay for urocanase activity, we characterized the inhibition. The inhibition was temperature-, concentration-, and time-dependent; 0.1, 0.5 and 1.0 mM glycylglycine inhibited the enzyme by 20%, 50% and 78%, respectively, in 60 min at 30 degrees C. Dithiothreitol and reduced glutathione did not prevent the process. The inhibition was a pseudo first-order reaction. Three ligands that bind to the active site, urocanate, imidazole-propionate (a competitive inhibitor) and sulfite, protected the enzyme from glycylglycine inhibition. The inhibition was very specific for glycylglycine, because fifteen related biochemicals, including glycine, triglycine, and tetraglycine, were not effective. Ethylenediaminetetraacetic acid and other chelators did not inhibit urocanase. Bovine liver urocanase was also inhibited by this peptide. The characteristics of this inhibition suggest that glycylglycine acts at the active site, does not function by metal binding and that minor alterations in the glycylglycine molecule preclude the inhibition. A specific inhibition of urocanases by glycylglycine has been observed.