PURIFICATION AND PROPERTIES OF THE FLAVOENZYME D-LACTATE DEHYDROGENASE FROM MEGASPHAERA-ELSDENII

A pyridine nucleotide independent d-lactate dehydrogenase has been purified to apparent homogeneity from the anaerobic bacterium Megasphaera elsdenii. The enzyme has a molecular weight of 105 000 by sedimentation equilibrium analysis with a subunit molecular weight of 55 000 by sodium dodecyl sulfate gel electrophoresis and is thus probably a dimer of identical subunits. It contains approximately 1 mol of FAD and 1 g-atom of Zn2+ per mol of protein subunit, and the flavin exhibits a fluorescence 1.7 times that of free FAD. An earlier purification [Brockman, H. L., & Wood, W. A. (1975) J. Bacteriol. 124, 1454-1461] results in substantial loss of the enzyme's zinc, which is required for catalytic activity. The new purification yields greater than 5 times the amount of enzyme previously isolated. The enzyme is specific for d-lactate, and no inhibition is observed with l-lactate. Surprisingly, the enzyme has a significant oxidase activity, which depends on the ionic strength. Vmax values of 190 and 530 min-1 were obtained at a Γ/2 of 0.224 and 0.442,respectively. Except for this atypically high oxygen reactivity, d-lactate dehydrogenase resembles other flavoenzyme dehydrogenases in that the flavin does not react with sulfite, the tryptophan content is low, and a neutral blue semiquinone is formed upon photochemical reduction. The enzyme flavin is reduced either by dithionite, by oxalate plus catalytic 5-deazaflavin in the presence of light, or by d-lactate. Two electrons per flavin were consumed in a dithionite titration, implying no other oxidation-reduction active groups. From equilibrations of the enzyme with varying ratios of d-lactate and pyruvate, an Em7 of -0.219 ± 0.007 V at 20 °C was calculated for the flavin. The enzyme requires dithiothreitol for stability. Rapid inactivtion results when the enzyme is incubated with a substoichiometric level of Cu2+. This inactivation can be reversed by dithiothreitol. It is proposed that the enzyme possesses a pair of cysteine residues capable of facile disulfide formation. © 1979, American Chemical Society. All rights reserved.