Mechanism of Action of the Flavoenzyme Lactate Oxidase

Abstract

Abstract Lactate oxidase from Mycobacterium smegmatis forms a spectrally detectable intermediate during reaction with each of the substrates, l-lactate, β-phenyl l-lactate, l-α-hydroxy-β-methylvalerate and l-α-hydroxyisovalerate. This intermediate, while present in greater amounts during anaerobic reactions, is also seen in the course of aerobic catalysis. The intermediate formed from each substrate has a molar extinction coefficient at 540 nm of about 800 m-1 cm-1 and its rate of formation, k2, and rate of reaction with oxygen, k4, are fast enough to account for turnover. A spectrally identical intermediate can be made by mixing reduced enzyme with an excess of the corresponding keto acid. The intermediate produced by mixing reduced enzyme and pyruvate, on letting in air, forms the normal catalytic product, acetate. Free hydrogen peroxide does not react with free pyruvate at the concentrations and in the short time interval in which the enzymatic reaction occurs, thus eliminating the possibility that the pathway for acetate production includes the nonenzymatic reaction of these two substances. We conclude that the intermediate is a charge transfer complex of reduced enzyme and keto acid and that it participates in catalysis. A mechanism and rate equation are presented in which product dissociation is the rate-determining step. All experiments were performed in 0.01 m imidazole-HCl buffer, pH 7.0, because anions such as phosphate are inhibitors which distort all spectral and kinetic studies.