Substrate-induced Conformational Changes of Liver Fructose 1,6-Diphosphatase Studied with the Hydrophobic Probe, 1-Anilinonaphthalene-8-sulfonate

The fluorescence emission of the hydrophobic probe, 1-anilinonaphthalene-8-sulfonate, was markedly increased in the presence of rabbit liver fructose 1,6-diphosphatase. Substrate produced a specific and reversible decrease in the fluorescence yield, which was linearly related to the amount of bound fructose-1,6-di-P. The fluorophore was very weakly bound to the phosphatase (Kdiss = 1.5 x 10-4 m). Addition of substrate did not change this binding constant but lowered the maximum emission yield. A very close agreement was found among the following constants for enzyme-substrate interaction: Km, Kdiss, and the substrate concentrations needed for half-maximal binding of AMP and for half-maximal fluorescence quenching. The fluorescence quenching of the enzyme-dye complex is further evidence for a substrate-induced change in protein conformation.