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| - Effects of Protein-Ligand Associations on the Subunit Interactions ofPhosphofructokinase from B. stearothermophilus
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| - Differences between the crystal structures of inhibitor-bound and uninhibited forms ofphosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allostericinhibition by phosphenolpyruvate (PEP) wherein a dimer−dimer interface within the tetrameric enzymeundergoes a quaternary shift. We have developed a labeling and hybridization technique to generate atetramer with subunits simultaneously containing two different extrinsic fluorophores in known subunitorientations. This construct has been utilized in the examination of the effects of allosteric ligand andsubstrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopictechniques including 2-photon, dual-channel fluorescence correlation spectroscopy (FCS). We demonstratethat PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface frombeyond the limits of experimental detection to nanomolar affinity, while conversely strengthening theinterface at which it is bound. The reduced interface affinity is specific to inhibitor binding because bindingthe 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.
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