Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue.
1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75-1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [(14)C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the (14)C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.
D001667:Binding, Competitive; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005486:Flavin Mononucleotide; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D007700:Kinetics; D007773:Lactates; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*
D005561:Formates; D007773:Lactates; D006639:Histidine; D005486:Flavin Mononucleotide; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D004047:Diethyl Pyrocarbonate