Influence of MgADP on phosphofructokinase from Escherichia coli. Elucidation of coupling interactions with both substrates. Academic Article uri icon

abstract

  • A comprehensive assessment is presented of the mutual influence that MgADP, MgATP, and fructose 6-phosphate (Fru-6-P) have on each other's binding to phosphofructokinase (PFK) from E. coli. When virtually any combination of these ligands binds to PFK it produces a significant perturbation in the intrinsic tryptophan fluorescence intensity and/or polarization which not only provides a means to follow binding in titration experiments but which also underscores the fact that more than two different enzyme conformations result from the binding of these ligands. When MgATP is saturating, the binding of MgADP to the allosteric site increases the affinity the enzyme subsequently displays for Fru-6-P. However, in the absence of MgATP, MgADP can bind to both the allosteric site and the nucleotide portion of the active site, with the latter antagonizing the binding of Fru-6-P to an extent that leads to an overall inhibition of Fru-6-P binding by MgADP. MgADP binding at the allosteric site also inhibits the binding of MgATP, indicating that under many circumstances MgADP should be more properly viewed as an inhibitor rather than an activator of E. coli PFK. After quantifying all of the 20 dissociation constants and 11 coupling parameters between ligand pairs pertinent to this three-ligand system, the more significant coupling parameters have been further characterized by examining their variation with temperature to establish the apparent enthalpy and entropy contributions to the corresponding coupling free energies. For both activating and inhibitory couplings, the enthalpy and entropy terms have the same sign as the coupling free energy.(ABSTRACT TRUNCATED AT 250 WORDS)

published proceedings

  • Biochemistry

author list (cited authors)

  • Johnson, J. L., & Reinhart, G. D

citation count

  • 26

complete list of authors

  • Johnson, JL||Reinhart, GD

publication date

  • March 1994