Equilibrium kinetic study of the mechanism of mitochondrial and supernatant malate dehydrogenases of bovine heart

Abstract

The kinetics at equilibrium have been examined for mitochondrial and supernatant malate dehydrogenases ( l-malate:NAD + oxidoreductase, EC 1.1.1.37) from bovine heart at catalytic concentration by means of isotopic exchange of reactants to compare their catalytic mechanisms. In accord with previous studies of lactate, alcohol and pig heart mitochondrial malate dehydrogenases the rate of substrate interchange exceeded the NAD + ⇌ NADH rate, in some cases by more than 100-fold, except at lowest substrate concentration. At pH 8.0 a marked depression in the NAD + ⇌ NADH rate to near zero without inhibition in the oxaloacetate ⇌ malate rate was compatible with a compulsory binding order with substrate binding to E (enzyme)-coenzyme complexes, but not to free enzyme. At pH 9.0 only a moderate depression in the NAD + ⇌ NADH rate was found, indicating a partially compulsory pathway in which coenzyme dissociation occurs from both E- coenzyme and E- coenzyme-substrate complexes, but is more rapid from the binary complex. Kinetics with variable concentration of non-reactive pairs of substrate and coenzyme suggested the formation of the abortive complex E- NADH malate, but not E- NAD +-oxaloacetate . The initial rate of reduction of oxaloacetate was in all cases greater than the equilibrium rates. Minimum estimates were obtained for some dissociation constants. Despite considerable structural differences, the similar kinetic behavior at equilibrium of the mitochondrial and supernatant enzymes suggests a uniform catalytic mechanism which includes a compulsory binding order at pH 8.0 with coenzyme binding prior to substrate, a partially compulsory pathway at pH 9.0, non-rate-limiting chemical transformation, and formation of the abortive complex, E- NADH-malate . This mechanism is also in accord with that previously found with pig heart mitochondrial malate dehydrogenase.