Studies on the reaction mechanism of lactate oxidase. Formation of two covalent flavin-substrate adducts on reaction with glycollate.
L-Lactate oxidase from Mycobacterium smegmatis catalyzes the oxidative decarboxylation of glycollate, with formate, CO2, and H2O as the major products. In addition, some "uncoupling" of the normal reaction occurs, with glyoxylate and H2O adition, some "uncoupling" of the normal reaction occurs, with glyoxylate and H2O2 as products. Glyoxylate is also a substrate (presumably as its hydrate); in this case, the reaction products are oxalate and H2O2. Evidence is presented that the enzyme recognizes glycollate as a prochiral substrate, differentiating between the Re- and Si-faces of the alpha carbon atom. Two highly fluorescent species are formed concomitantly from the reaction with glycollate; they are proposed to be covalent alpha-glycollyl adducts to the reduced flavin position N(5). One of these adducts is labile and in rapid equilibrium with oxidized enzyme and glycollate, and with the complex of reduced enzyme and glyoxylate; this adduct is a catalytically competent intermediate. The other adduct is comparatively stable (t 1/2 for decay = 20 min at 25 degrees C) and does not react with O2. It is formed at a rate approximately 1% that of the catalytic adduct, but because of its lack of reaction with O2 and its stability, it gradually accumulates during catalytic turnover, resulting in catalytically incompetent enzyme. An isotope effect of approximately 4 is found in the reduction of oxidized enzyme flavin and in the formation of the labile fluorescent adduct, when alpha-2H2-glycollate or (R)-glycollate-2-d is used, but not with the (S)-glycollate-2-d enantiomer. It is concluded that the catalytic adduct is formed by hydrogen abstraction from the Re-face of glycollate.
D000332:Aerobiosis; D000693:Anaerobiosis; D003903:Deuterium; D005415:Flavins; D006016:Glycolates; D006038:Glyoxylates; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009161:Mycobacterium / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D005415:Flavins; D006016:Glycolates; D006038:Glyoxylates; D003903:Deuterium; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C021497:lactate 2-monooxygenase