Substrate-mediated inactivation of urocanase from Pseudomonas putida. Evidence for an essential sulfhydryl group.
Incubation of urocanase from Pseudomonas putida with either its substrate, urocanic acid, or product, 4'(5')-imidazolone-5'(4')-propionic acid, resulted in an oxygen-dependent inhibition of enzyme activity. Coincident with the inactivation was the stoichiometric incorporation of radioactivity from [14C]urocanate into the protein. NAD+ which is required for activity or urocanase was not directly involved in the inactivation process. The inactivation of urocanase was irreversible, could be partially blocked by the competitive inhibitor imidazolepropionate, and involved the modification of a single active-site thiol. The inhibition resulted from oxidative decomposition of 4'(5')-imidazolone-5'(4')-propionate but was not due to the formation of the major degradative product, 4-ketoglutaramate, since this compound was not an irreversible inactivator of urocanase although it did produce some inhibition at high concentrations. A mechanism is presented in which a reactive imine intermediate in the decomposition scheme is subject to nucleophilic attack by an active-site thiol, thereby generating a covalent enzyme--thioaminal adduct. These results emphasize the importance of a catalytic center sulfhydryl group for urocanase activity.
D000332:Aerobiosis; D000693:Anaerobiosis; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors*; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D009243:NAD / Q000494:pharmacology; D011422:Propionates / Q000494:pharmacology; D011549:Pseudomonas / Q000201:enzymology*; D013438:Sulfhydryl Compounds; D014559:Urocanate Hydratase / Q000037:antagonists & inhibitors*; D014560:Urocanic Acid / Q000494:pharmacology*
D007093:Imidazoles; D011422:Propionates; D013438:Sulfhydryl Compounds; D009243:NAD; C018977:imidazol-4-one-5-propionic acid; D004228:Dithionitrobenzoic Acid; D006836:Hydro-Lyases; D014559:Urocanate Hydratase; D014560:Urocanic Acid; D004229:Dithiothreitol