Inactivation of L-lactate monooxygenase with 2,3-butanedione and phenylglyoxal.
L-Lactate monooxygenase from Mycobacterium phlei is inactivated by reaction either with 2,3-butanedione in borate or in 2,6-lutidine buffer or with phenyglyoxal in 2,6-lutidine buffer. The activation with 2.3 butanedione in borate buffer is irreversible in the presence of excess borate, but essentially complete recovery of activity occurs on exchange of phosphate for borate buffer. In 50 mM borate, inactivation with 2,3-butanedione exhibits saturation kinetics with respect to increasing concentrations of 2,3-butanedione, whereas second-order kinetics for inactivation are seen in 200 mM borate. In 2.6-lutidine buffer, the inactivation is rapid, irreversible on change of buffer, and second order overall. Complete inactivation of the enzyme by phenylglyoxal in 2,6-lutidine buffer occurs on incorporation of 2 equiv of phenylglyoxal per subunit, but only one arginyl residue per subunit is modified. The inactivation is irreversible and second order in phenyglyoxal. There is substantial protection from inactivation in the presence of D-lactate, a competitive inhibitor of the enzyme. It is suggested that an arginyl residue in the active site in L-lactate monooxygenase is involved in the binding of the carboxyl group of substrates to the enzyme. An explanation for the unusual kinetics of inactivation with 2,3-butanedione in borate and with phenylglyoxal in 2,6-lutidine is offered.
D000447:Aldehydes / Q000494:pharmacology*; D002074:Butanones / Q000494:pharmacology*; D003931:Diacetyl / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D009168:Mycobacterium phlei / Q000201:enzymology; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D010658:Phenylglyoxal / Q000494:pharmacology*
D000447:Aldehydes; D002074:Butanones; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C021497:lactate 2-monooxygenase; D003931:Diacetyl; D010658:Phenylglyoxal