title: Pre-steady state quantification of the allosteric influence of Escherichia coli phosphofructokinase.
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.
authors.affiliation: Division of Pathology and Laboratory Medicine, the University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA. aspham@mail.mdanderson.org
authors.forename: A S
authors.initials: AS
authors.lastname: Pham
authors.forename: G D
authors.initials: GD
authors.lastname: Reinhart
mesh_terms: D000494:Allosteric Regulation; D004926:Escherichia coli / Q000201:enzymology*; D005636:Fructosephosphates / Q000378:metabolism; D007700:Kinetics; D010728:Phosphoenolpyruvate / Q000378:metabolism; D010732:Phosphofructokinase-1 / Q000737:chemistry / Q000378:metabolism*
chemical_list: D005636:Fructosephosphates; C027618:fructose-6-phosphate; D010728:Phosphoenolpyruvate; D010732:Phosphofructokinase-1