Effects of protein-ligand associations on the subunit interactions of phosphofructokinase from B. stearothermophilus.
Differences between the crystal structures of inhibitor-bound and uninhibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphoenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits simultaneously containing two different extrinsic fluorophores in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel fluorescence correlation spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor binding because binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated.
Quinlan
R Jason
RJ
Department of Biochemistry and Biophysics, Texas A&M University and the Texas Agricultural Experiment Station, College Station, Texas 77843-2128, USA.
Reinhart
Gregory D
GD
D016880:Anisotropy; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D025221:Phosphofructokinases / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D008024:Ligands; D021122:Protein Subunits; D025221:Phosphofructokinases