Fluorescence energy-transfer measurements between coenzyme A and flavin adenine dinucleotide binding sites of the Escherichia coli pyruvate dehydrogenase multienzyme complex

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Abstract
The interaction of the pyruvate dehydrogenase multienzyme complex from E. coli with 1,N6-etheno-CoA (.epsilon.CoA) and CoA was 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 is given. Direct binding studies with .epsilon.CoA indicate that 25 bound .epsilon.CoA molecules/multienzyme complex can be readily displaced by CoA, while approximately 21 bound .epsilon.CoA molecules/transacetylase-flavoprotein subcomplex can be displaced by CoA. The Kd for the CoA displaceable .epsilon.CoA is 57.8 .mu.M for the complex and 126 .mu.M for the subcomplex in 0.02 M potassium phosphate (pH 7.0) at 5.degree. C. The kinetic behavior of .epsilon.CoA as a substrate was investigated and compared with CoA under a variety of conditions; the apparent Km for .epsilon.CoA are considerably larger than those for CoA, while the corresponding Vmax are smaller. Fluorescence energy transfer measurements between bound .epsilon.CoA on the dihydrolipoyl transacetylase and FAR on the dihydrolipoyl dehydrogenase in the complex or subcomplex indicate, assuming the emission and absorption dipoles are randomly oriented, that these 2 probes must be at least 50 .ANG. apart.