Studies on the kinetic mechanism of lipoamide dehydrogenase from rat liver mitochondria.

The kinetic mechanism of lipoamide dehydrogenase has been studied at pH 8.0, 37”, using the enzyme from rat liver mitochondria. Initial velocity patterns obtained for both the forward and reverse reactions were a series of parallel lines. Michaelis constants for the reactants are: NAD, 0.52 mu; dihydrolipoamide, 0.49 mu; NADH, 0.062 mM; lipoamide, 0.84 mu. Isotopic exchange between NAD and NADH was also studied. The exchange rate in the absence of lipoyl derivatives was not significantly increased by the presence of all of the reactants. These data, as well as the results from product inhibition studies, are consistent with a Bi Bi Ping Pong mechanism for this enzyme as originally proposed by Massey et al. (MASSEY, V., GIBSON, Q. H., AND VEEGER, C. (1960) Biochem. J. 77, 341). In addition, the formation of kinetically significant abortive complexes between the enzyme and NADH or lipoamide is shown.