Studies on the Reaction Mechanism of Lactate Oxidase : Formation of two covalent Flavin-Substrate adducts on reaction with Glycolate

Abstract

L-Lactate oxidase from Mycobacterium smegmatis catalyzes the oxidative decarboxylation of glycollate, with formate, CO2, and H20 as the major products. In addition, some uncoupling of the normal reaction occurs, with glyoxylate and H202 as products. Glyoxylate is also a substrate( presumably as its hydrate); in this case, the reaction products are oxalate and H202. Evidence is presented that the enzyme recognizes glycollate as a prochiral substrate, differentiating between the Re- and Si-faces of the α carbon atom. Two highly fluorescent species are formed concomitantly from the reaction with glycollate; they are proposed to be covalent α-glycollyl adducts to the reduced flavin position N(5). One of these adducts is liabile 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 (t1/2 for decay = 20 min at 25°C) and does not react with 02. 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 ~4 is found in the reduction of oxidized enzyme flavin and in the formation of the labile fluorescent adduct, when α-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.