PMID:11300796
| Citation |
Pham, AS and Reinhart, GD (2001) MgATP-dependent activation by phosphoenolpyruvate of the E187A mutant of Escherichia coli phosphofructokinase. Biochemistry 40:4150-8 |
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| Abstract |
Using enzymatic assays and steady-state fluorescence emission, we performed a linkage analysis of the three-ligand interaction of fructose 6-phosphate (Fru-6-P), phosphoenolpyruvate (PEP), and MgATP on E187A mutant Escherichia coli phosphofructokinase (PFK). PEP allosterically inhibits Fru-6-P binding to E. coli PFK. The magnitude of antagonism is 90-fold in the absence and 60-fold in the presence of a saturating concentration of MgATP [Johnson, J. J., and Reinhart, G. D. (1997) Biochemistry 36, 12814-12822]. Substituting an alanine for the glutamate at position 187, located in the allosteric site (i.e., mutant E187A), activates Fru-6-P binding and inhibits the maximal rate of enzyme turnover [Lau, F. T.-K., and Fersht, A. R. (1987) Nature 326, 811-812]. The allosteric action of PEP appears to depend on the presence of the cosubstrate MgATP. In the presence of a saturating concentration of MgATP, PEP enhances the binding of Fru-6-P to the enzyme by a modest 2-fold. Decreasing the concentration of MgATP mitigates the extent of activation. At MgATP concentrations approaching 25 microM, PEP becomes insensitive to the binding of Fru-6-P. At MgATP concentrations < 25 microM, PEP "crosses over" and becomes antagonistic toward substrate binding. The present study examines the role of Glu 187 at the allosteric site in the binding of Fru-6-P and offers a more complex explanation of the mechanism than that described by traditional allosteric mechanistic models. |
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| Keywords |
Adenosine Triphosphate/chemistry; Adenosine Triphosphate/metabolism; Alanine/genetics; Binding Sites/genetics; Entropy; Enzyme Activation/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Fructosephosphates/metabolism; Glutamic Acid/genetics; Kinetics; Ligands; Mutagenesis, Site-Directed; Phosphoenolpyruvate/chemistry; Phosphofructokinase-1/genetics; Phosphofructokinase-1/metabolism; Spectrometry, Fluorescence; Temperature; Thermodynamics |
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