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.