Fluorescence energy transfer between heterologous active sites of affinity-labeled aspartokinase of Escherichia coli.
The distance between aspartokinase and homoserine dehydrogenase active sites was determined using fluorescence energy transfer between modified substrates. The fluorescent 1,N(6)-ethenoadenosine 5'-triphosphate was bound at the kinase active site by Co(III) affinity labeling. Reduced thionicotinamide adenine dinucleotide phosphate quenched the fluorescence of bound nucleotide. Fluorescence depolarization measurements led to a delimitation of the value of the dipolar orientation factor to the range 0.3 to 2.8. The distance between the fluorescent probe and the quencher was 29 +/- 4 A. In the presence of threonine, this distance increased to 36 +/- 5 A. Threonine binding either increased the intersite distance by ca. 7 A or caused a reorientation of the probe at the dehydrogenase site.
D000345:Affinity Labels / Q000378:metabolism*; D001222:Aspartate Kinase / Q000378:metabolism; D001665:Binding Sites; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology*; D006715:Homoserine Dehydrogenase / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D009097:Multienzyme Complexes / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D000345:Affinity Labels; D009097:Multienzyme Complexes; D006715:Homoserine Dehydrogenase; D010770:Phosphotransferases; D001222:Aspartate Kinase