Molecular mechanism of regulation of the pyruvate dehydrogenase complex from E. coli.
The pyruvate dehydrogenase multienzyme complex from E. coli shows a sigmoidal dependency of the reaction rate on the substrate concentration when product formation is followed in the presence of physiological concentrations of the cofactor thiamin diphosphate. To elucidate the molecular mechanism of this regulation, the influence of the substrate pyruvate on the coenzyme-protein interaction has been investigated using several coenzyme analogues. The observed binding constants of all coenzymatically active analogues are increased in the presence of the substrate pyruvate, whereas those of all coenzymatically inactive analogues are not altered in the presence of pyruvate. This points to an increased binding affinity of a reaction-intermediate-coenzyme complex to the protein. Since cofactor binding and dissociation at physiological concentrations of thiamin diphosphate are slow compared to the catalytic reaction, a slow transition to the active state of the enzyme occurs. After lowering the pyruvate concentration, the opposite effect, a dissociation of the thiamin diphosphate from the enzyme is observed. This slow substrate dependent enhancement of cofactor binding enables efficient regulation of the pyruvate dehydrogenase complex by its substrate pyruvate.
Hennig
J
J
Martin-Luther-Universität Halle-Wittenberg, Institut für Biochemie, Kurt-Mothes-Strasse 3, D-06099 Halle, Germany.
Kern
G
G
Neef
H
H
Spinka
M
M
Bisswanger
H
H
Hübner
G
G
D003067:Coenzymes / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D011485:Protein Binding; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013835:Thiamine Pyrophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism
D003067:Coenzymes; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D019289:Pyruvic Acid; D014364:Tryptophan; D004047:Diethyl Pyrocarbonate; D013835:Thiamine Pyrophosphate