Fluorescence energy transfer between heterologous active sites of affinity-labeled aspartokinase of Escherichia coli

Abstract

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.

Distances among coenzyme and metal sites of NADP+-dependent isocitrate dehydrogenase using resonance energy transfer

“…TNADPH was synthesized from TNADP4" by enzymatic reduction using glucose-6-phosphate dehydrogenase (Wright & Takahashi, 1977). To a 1-mL solution containing 2.6 mM TNADP4", 50 mM glucose 6-phosphate (Sigma), and 50 mM Tris-HCl, pH 8.0, was added glucose-6-phosphate dehydrogenase to a final concentration of 21 ^g/mL.…”

The orientational freedom of molecular probes. The orientation factor in intramolecular energy transfer

“…In the 30 years or so since Frster developed an exact quantum mechanical theory of resonance energy transfer for the "very weak" dipole-dipole coupling limit (Frster, 1948(Frster, , 1951(Frster, , 1965, an extensive and still rapidly growing literature has described the utilization of the phenomenon to determine intramolecular separations (e.g., recently Wu et al, 1976;Langlois et al, 1976;Shepherd et al, 1976;Wright and Takahashi, 1977;Papadakis and Hammes, 1977;Zukin et al, 1977) and conformational dynamics (Haas et al, 1975(Haas et al, , 1977Ohmine et al, 1977) of mainly biological macromolecules. Further impetus to such studies was undoubtedly provided by the finding that the predicted inverse sixth-power distance dependence of the transfer rate holds closely for several series of oligomers having donor (D) and acceptor (A) moieties bound covalently at their ends, the separation of which depends on the number of intermediate monomer units (Stryer and Haugland, 1967;Conrad and Brand, 1968;Gabor, 1968).…”

Glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides. Conformational transitions induced by NAD, NADP and glucose 6-phosphate monitored by fluorescent probes

“…Native Enzyme Monitored by S-NADPH Fluorescence. The corrected emission spectrum of S-NADPH was reported by Wright & Takahashi (1977). Uncorrected spectra show excitation and emission maxima at 399 and 490 nm, respectively (Figure 4).…”