Studies on the catalytic mechanism of lactate oxidase : formation of enantiometric flavin-N(5)-glycollyl adducts via carbanion intermediates

Abstract

L-Lactate oxidase from Mycobacterium smegmatis reacts with the prochiral subsgtlryactoel late and forms a labile, catalytically competent glycollyl adduct in addition to a similar, but comparatively stable adduct (Massey, V., Ghisla, S., and Kieschke, K. (1980) J. Bid. Chem 255,2796-2806). The latter was isolated by Sephadex G-25 chromatography at 0-4°C and was also obtained from lactate oxidase, in which FMN had been replaced by the analogue 2-thio-FMN. The stable adduct is identical with the product obtained from illumination of the lactate oxidase●tartronate complex (Ghisla, S., Massey, V., and Choong, Y. S. (1979) J. Biol. Chem 254, 10662-10669) and thus has the structure of a glycollyl adduct to position N(5) of the reduced enzyme flavin. The stable adduct decays directly to oxidized enzyme and glycollate, with a t1/2 of 20 min at 25°C; the Arrhenius activation energy 21.4 kcal/mol. When the adduct is formed from reaction with [2,S- 2H] or with α-dideuteroglycollate, the decay reaction shows an isotope effect of 1.5. In contrast, no isotope effect is observed when the adduct is obtained from [2,R-2H]glycollate. Using [2,R-3H]- and [2,S-3H]glycollate, it is shown that the enzyme oxidizes catalytically the Re-hydrogen of this substrate, which is stereochemically equivalent to the α-hydrogen of L-lactate. On decay of the adduct, the Si-hydrogen bond of glycollate is (re)formed. This is demonstrated by the stereochemistry of glycollate obtained from decay of adduct formed photochemically from enzyme and [2-3H]- tartronate. The direct formation of a covalent glycollyl adduct at position N(5) of reduced FMN is interpreted as being equivalent to addition of a transient carbanion, which is formed by abstraction of a proton from the glycollate α position.