Beef Heart Malic Dehydrogenases

The stoichiometry of reduced diphosphopyridine nucleotide binding to supernatant malic dehydrogenase (S-MDH) was determined by fluorometric techniques and a Sephadex gel filtration procedure. Both types of measurement yielded a stoichiometry of 1 mole of DPNH bound per mole of enzyme. This stoichiometry was unaffected by the presence of d-malate, a compound which is known to form a ternary enzyme complex having distinctive fluorescent properties. The dissociation constants for the binary enzyme-DPNH and ternary enzyme-DPNH-d-malate complexes with respect to DPNH were determined. Several criteria were used to determine the effectiveness of a number of compounds in forming ternary complexes with S-MDH and DPNH. A requisite for ternary complex formation was a dicarboxylic acid with chain length of 3 to 4 carbon atoms. Although an α-hydroxyl group was not necessary for ternary complex formation, the introduction of such a group in a specific steric configuration resulted in a loss of capacity to form such a complex. Formation of strong fluorescing complexes by a number of dicarboxylic acids is not necessarily related to their effectiveness as inhibitors of S-MDH activity. The presence of an α-hydroxyl group appears to be required for inhibition, although added steric considerations involving the β carbon atom may obliterate such inhibitory capacity.