Dihydrolipoamide dehydrogenase component of the pyruvate dehydrogenase complex from Escherichia coli K12. Comparative characterization of the free and the complex-bound component.
The regulation of the biosynthesis of dihydrolipoamide dehydrogenase is dependent on the biosynthesis of the pyruvate dehydrogenase complex. The gene coding for the dihydrolipoamide dehydrogenase appears to be included in the regulation of the pyruvate dehydrogenase operon. Possibly a secondary promoter is inserted. Dihydrolipoamide dehydrogenase was purified in its free form using a dihydrolipoamide-agarose affinity column and avoiding denaturating conditions. The enzyme shows complete cross-reactivity with antibodies against the pyruvate dehydrogenase complex and has a higher specific activity than any preparations described thus far. The transition state activation energy of the catalytic activity is smaller for the complex-bound enzyme than that found for the free enzyme. In its complexed form, the enzyme also proves to be somewhat more stable under alkaline conditions. The reaction catalyzed by the dihydrolipoamide dehydrogenase shows the behaviour of a ping-pong mechanism. The Michaelis constants for the substrates NAD and dihydrolipoamide found with the free enzyme are about four times those observed with the enzyme integrated into the native complex. The catalytic reaction of both forms of the dihydrolipoamide dehydrogenase is inhibited by NADH. The mechanism of this inhibition cannot simply be explained by a product inhibition. Rather the further reduction of the catalytically active, half-reduced enzyme form to the catalytically inactive, fully reduced form has to be considered as causing the inhibition.
D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D007106:Immune Sera; D007118:Immunoassay; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D013053:Spectrophotometry
D007106:Immune Sera; D046911:Macromolecular Substances; D011768:Pyruvate Dehydrogenase Complex; D008058:Dihydrolipoamide Dehydrogenase