Inactivation and modification of lactate oxidase with fluorodinitrobenzene.
1. Dinitrophenylation of 2 +/- 0.2mol of residues/mol of enzyme-bound FMN resulted in the complete inactivation of the flavoenzyme L-lactate oxidase. 2. Hydrolysates of the inactivated enzyme contained 1mol each of Nim-Dnp-histidine (abbreviation: Dnp-,2,4-dinitrophenyl-; Nim indicates that either of the N atoms in the imidazole ring is substituted) and epsilon-Dnp-lysine/mol of FMN. 3. Competitive inhibitors decreased the extent of inactivation to a 10% loss of activity, and dinitrophenylation was decreased from 2 to approx. 0.5mol/mol of FMN. Only Nim-Dnp-histidine was detected in the hydrolysates. 4. Although the dinitrophenylated enzyme did not possess enzyme activitiy, L-lactate reduced approx. 50% of the enzyme-bound flavin slowly (0.6min-1), and approx. 50% of the flavin in the modified enzyme-bound flavin slowly (0.6min-1), and approx. 50% of the flavin in the modified enzyme formed a complex with bisulphite. 6. The modified enzyme (2mol of Dnp/mol of FMN) was unable to bind substrate analogues and competitive inhibitors.
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D004139:Dinitrofluorobenzene / Q000494:pharmacology*; D007700:Kinetics; D007773:Lactates; D008314:Malonates; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D009566:Nitrates; D009578:Nitrobenzenes / Q000494:pharmacology*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D010710:Phosphates; D013447:Sulfites
D000596:Amino Acids; D007773:Lactates; D008314:Malonates; D009566:Nitrates; D009578:Nitrobenzenes; D010710:Phosphates; D013447:Sulfites; D004139:Dinitrofluorobenzene; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C021497:lactate 2-monooxygenase