Fluorescence energy transfer measurements between ligand binding sites of the pyruvate dehydrogenase multienzyme complex.
The interaction of the pyruvate dehydrogenase multienzyme complex from Escherichia coli with 8-anilino-1-naphthalenesulfonate (ANS), pyruvate, and acetyl-CoA has been investigated using equilibrium binding, steady-state fluorescence, and fluorescence lifetime measurements. The fluorescnece of ANS is greatly enhanced when bound to the enzyme complex and to the pyruvate dehydrogenase component of the complex. Approximately 22 molecules of ANS are bound to a molecule of the complex with a binding constant of 3.69 muM in 0.03 M potassium potassium phosphate (pH 7.0). Direct and competitive binding measurements indicate that about 42 pyruvate binding sites are present per mole of enzyme complex which has been stripped of thiamine diphosphate; the number of binding sites is reduced to 28,5 in the presence of a saturating concentration of thiochrome diphosphate, a thiamine diphosphate analogue. The dissociation constant for pyruvate to the enzyme complex in the presence of thiochrome diphosphate is 308 muM in 0.02 M potassium phosphate (pH 7.0). Pyruvate, thiochrome diphosphate, and acetyl-CoA all displace ANS from the enzyme complex. In the cases of pyruvate and thiochrome diphosphate, the concentration dependence of the displacements suggests the displacement is allosteric, while in the case of acetyl-CoA direct competition appears to be involved. GTP decreased the effect of acetyl-CoA to the enzyme complex indicate that 24-26 bound acetyl-CoA molecules per complex can be readily displaced by ANS, and the binding of acetyl-CoA to these sites displays positive cooperativity. Fluorescence energy transfer measurements between bound ANS on the pyruvate dehydrogenase enzyme and FAD on the dihydrolipoyl dehydrogenase enzyme indicate, assuming the emission and absorption dipoles are randomly oriented, that these two probes must be at least 58 A apart in the intact complex.
D000817:Anilino Naphthalenesulfonates; D001665:Binding Sites; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology; D008433:Mathematics; D011485:Protein Binding; D011768:Pyruvate Dehydrogenase Complex; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; D011768:Pyruvate Dehydrogenase Complex