Fluorescence energy-transfer measurements between coenzyme A and flavin adenine dinucleotide binding sites of the Escherichia coli pyruvate dehydrogenase multienzyme complex.
The interaction of the pyruvate dehydrogenase multienzyme complex from Escherichia coli with 1,N6-etheno-CoA (epsilonCoA) and coenzyme A (CoA) has been investigated using equilibrium binding, steady-state fluorescence, and fluorescence lifetime measurements. A procedure for the resolution of the pyruvate dehydrogenase multienzyme complex into the pyruvate dehydrogenase enzyme and the transacetylase-flavoprotein subcomplex also is given. Direct binding studies with epsilonCoA indicate that 25 bound epsilonCoA molecules/multienzyme complex can be readily displaced by CoA, while approximately 21 bound epsilonCoA molecules/transacetylase-flavoprotein subcomplex can be displaced by CoA. The dissociation constant for the CoA displaceable epsilonCoA is 57.8 muM for the complex and 126 muM for the subcomplex in 0.02 M potassium phosphate (pH 7.0) at 5 degrees C. The kinetic behavior of epsilonCoA as a substrate was investigated and compared with that of CoA under a variety of conditions; the apparent Michaelis constants for epsilonCoA are considerably larger than those for CoA, while the corresponding maximal velocities are smaller. Fluorescence energy transfer measurements between bound epsilonCoA on the dihydrolipoyl transacetylase enzyme and flavin adenine dinucleotide on the dihydrolipoyl dehydrogenase enzyme either in the complex or subcomplex indicate, assuming the emission and absorption dipoles are randomly oriented, that these two probes must be at least 50 A apart.
D001051:Apoenzymes; D001665:Binding Sites; D003065:Coenzyme A / Q000494:pharmacology; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000494:pharmacology; D007700:Kinetics; D008433:Mathematics; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D001051:Apoenzymes; D005182:Flavin-Adenine Dinucleotide; D003065:Coenzyme A