Evaluation of ligand interactions of Bacillus stearothermophilus phosphofructokinase using the fluorescence of a tryptophan-shift mutant.
Conference Paper
Overview
Additional Document Info
View All
Overview
abstract
Phosphofructokinase (PFK) from B. Stearothermophilus is activated by MgADP and inhibited by phospho(enol)pyruvate (PEP). This enzyme is a homotetramer containing one tryptophan per subunit. The fluorescence of the tryptophan in its native position is unresponsive to ligand interactions. Site-directed mutagenesis has been used to generate a Rtryptophan-shiftedS double mutant in which the tryptophan (position 179) has been changed to a phenylalanine and the phenylalanine (position 230) has been changed to a tryptophan. The mutation occurs on the side directly opposite of the native tryptophan on the small domain of the subunit. According to the x-ray crystal structure of the fructose-6-phosphate and MgADP-bound wild type enzyme, the spatial distance of the wild type tryptophan and the mutated tryptophan from the allosteric ADP binding site is approximately the same and equal to 25 Angstroms. The mutant enzyme has similar kinetic properties to wild type, and the fluorescence of the mutant is responsive to ligand binding. PEP causes a 22% increase in fluorescence intensity and a polarization increase of 0.015. Using these fluorescence changes, a dissociation constant of 5.3 1 0.2 fiM has been measured. This value compares favorably to the dissociation constant of 4.6 1 0.4 /iM that has been estimated by kinetic analysis. MgADP causes a 20% decrease in fluorescence intensity and no change is detected in polarization. The dissociation constant for MgADP is 15 1 9 M.
