Investigation of the anticooperative binding of NADH to porcine heart mitochondrial malate dehydrogenase.

Recent studies in this laboratory have shown that porcine heart mitochondrial malate dehydrogenase (Lmalate:NAD* oxidoreductase, EC 1.1.1.37) exhibits both a concentration and a pH-dependent dissociation (Bleile, D. M., Schulz, R. A., Gregory, E. M., and Harrison, J. H. (1977) J. Biol. Chem. 252,756758; Hodges, C. T., Wiggins, J. C., and Harrison, J. H. (1977) J. BioZ. Chem. 252, 6038-6041). Since previous studies on the binding of NADH to this enzyme (Holbrook, J. J., and Wolfe, R. G. (1972) Biochemistry 11, 2499-2502) were performed under conditions now known to favor the dissociation of this enzyme, the binding of NADH to malate dehydrogenase has been reinvestigated. Additional binding studies were performed both in the presence and absence of the substrate L-malate and the inhibitor hydroxymalonate. The binding of NADH to malate dehydrogenase at pH 7.5 in the absence of substrates or inhibitors was found to be anticooperative with apparent dissociation constants KI and Ka of 0.45 pm and 1.7 PM, respectively. In the presence of hydroxymalonate, NADH binding is also anticooperative with an apparent K1 of 0.02 pM and an apparent KZ of 0.20 PM. This anticooperative nature of NADH binding is not observed in the presence of L-malate and a single apparent dissociation constant of 1.6 pM is exhibited. When malate dehydrogenase is titrated at pH 5.0 with NADH, conditions under which the enzyme is known to be dissociated, the anticooperative binding is also abolished. The presence of anticooperative binding of NADH seems to support the compulsory ordered reciprocating mechanism previously proposed for this enzyme (Harada, K., and Wolfe, R. G. (1968) J. Biol. Chem 243,4131-4137).