The Membrane-Bound l- and d-Lactate Dehydrogenase Activities in Mitochondria from Euglena gracilis

The activity of the pyridine nucleotide-independent lactate dehydrogenase (iLDH) was characterized in mitochondria isolated from the protist Euglena gracilis. The dissociation constants for l- and d-lactate were similar, but the Vmax was higher with the d isomer. A ping-pong kinetic mechanism was displayed with 2,4-dichlorophenol-indolphenol (DCPIP), or coenzyme Q1, reacting as the second substrate with the modified, reduced enzyme. Oxamate was a competitive inhibitor against both l- and d-lactate. Oxalate exerted a mixed-type inhibition regarding l- or d-lactate and also against DCPIP. The rate of l-lactate uptake was partially inhibited by mersalyl and lower than the rate of dehydrogenation, which was mersalyl-insensitive. These data suggested that the active site of l-iLDH was orientated toward the intermembrane space. The following observations indicated the existence of two stereo-specific iLDH enzymes in the inner membrane of Euglena mitochondria: a greater affinity of the d-iLDH for both inhibitors, d-iLDH thermo-stability at 70°C and denaturation of l-iLDH, opposite signs in the enthalpy change for the association reaction of the isomers to the enzyme, differential solubilization of both activities with detergents, and different molecular mass.