MgATP-dependent activation by phosphoenolpyruvate of the E187A mutant of Escherichia coli phosphofructokinase.
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.
Pham
A S
AS
Division of Pathology and Laboratory Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. aspham@mail.mdanderson.org
Reinhart
G D
GD
D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000409:Alanine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D019277:Entropy; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005636:Fructosephosphates / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D010728:Phosphoenolpyruvate / Q000737:chemistry*; D010732:Phosphofructokinase-1 / Q000235:genetics* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D005636:Fructosephosphates; D008024:Ligands; D018698:Glutamic Acid; C027618:fructose-6-phosphate; D010728:Phosphoenolpyruvate; D000255:Adenosine Triphosphate; D010732:Phosphofructokinase-1; D000409:Alanine