Kinetic studies on the inactivation of L-lactate oxidase by [the acetylenic suicide substrate] 2-hydroxy-3-butynoate.
2-Hydroxy-3-butynoate is both a substrate and an irreversible inactivator of the flavoenzyme L-lactate oxidase. The partitioning between catalytic oxidation of 2-hydroxy-3-butynoate and inactivation of the enzyme is determined by the concentration of the second substrate, O2. Rapid reaction studies show the formation of an intermediate which is common to both the oxidation and inactivation pathways. This intermediate appears to be a charge-transfer complex between enzyme-reduced flavin and 2-keto-3-butynoate. It is characterized by a long-wavelength absorbing band (gamma(max) 600 nm) and lack of fluorescence, making it easily distinguished from the subsequently formed inactivated enzyme, which has no long wavelength absorption (gamma(max) 318, 368 nm) and which is strongly fluorescent. Inactivation is also accomplished by reaction of the reduced enzyme with 2-keto-3-butynoate. The absorbance and fluorescence characteristics of the inactivated enzyme are similar to those of a model compound, C(4a), N(5)-propano-bridged FMN bound to apolactate oxidase. That the modified chromophore of the inactivated enzyme is an adduct involving both the C(4a) and N5 positions is further supported by the spectral and fluorescence changes resulting from treatment of the inactivated enzyme with borohydride.
D000693:Anaerobiosis; D005182:Flavin-Adenine Dinucleotide; D006885:Hydroxybutyrates / Q000494:pharmacology*; D007700:Kinetics; D007773:Lactates; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors*; D009161:Mycobacterium / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors*; D010101:Oxygen Consumption; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
D006885:Hydroxybutyrates; D007773:Lactates; D005182:Flavin-Adenine Dinucleotide; C002479:2-hydroxy-3-butynoic acid; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C021497:lactate 2-monooxygenase