PMID:11443117
| Citation |
Pham, AS and Reinhart, GD (2001) Pre-steady state quantification of the allosteric influence of Escherichia coli phosphofructokinase. J. Biol. Chem. 276:34388-95 |
|---|---|
| Abstract |
Stopped-flow kinetics was utilized to determine how allosteric activators and inhibitors of wild-type Escherichia coli phosphofructokinase influenced the kinetic rate and equilibrium constants of the binding of substrate fructose 6-phosphate. Monitoring pre-steady state fluorescence intensity emission changes upon an addition of a ligand to the enzyme was possible by a unique tryptophan per subunit of the enzyme. Binding of fructose 6-phosphate to the enzyme displayed a two-step process, with a fast complex formation step followed by a relatively slower isomerization step. Systematic addition of fructose 6-phosphate to phosphofructokinase in the absence and presence of several fixed concentrations of phosphoenolpyruvate indicated that the inhibitor binds to the enzyme concurrently with the substrate, forming a ternary complex and inducing a conformational change, rather than a displacement of the equilibrium as predicted by the classical two-state model (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118). The activator, MgADP, also altered the affinity of fructose 6-phosphate to the enzyme by forming a ternary complex. Furthermore, both phosphoenolpyruvate and MgADP act by influencing the fast complex formation step while leaving the slower enzyme isomerization step essentially unchanged. |
| Links |
PubMed Online version:10.1074/jbc.M102785200 |
| Keywords |
Allosteric Regulation; Escherichia coli/enzymology; Fructosephosphates/metabolism; Kinetics; Phosphoenolpyruvate/metabolism; Phosphofructokinase-1/chemistry; Phosphofructokinase-1/metabolism |
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