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.
Highlights
Phosphofructokinase (PFK)1 catalyzes the phosphorylation of fructose 6-phosphate (Fru-6-P) from MgATP to form fructose 1,6-bisphosphate and MgADP
Binding of Fru-6-P to the PEP1⁄7PFK Complex—To determine how allosteric ligands affect the rate constant for binding, we monitored Fru-6-P binding in a range of fixed concentrations of PEP preincubated with the enzyme
Kinetic Scheme Modification—The results indicate that the single-substrate, single-effector thermodynamic box previously described by Reinhart [28] must be expanded to account for the intermediate isomerization step reflecting the interaction of Fru-6-P with the enzyme
Summary
The purification protocol and steady state kinetics were performed as described previously [18, 19]. No dependence of the observed rate constant on PFK concentration was detected within this concentration range. Values of kobs as a function of ligand concentrations were fit to the rate-dependent mechanisms described below. As with steady state conditions, experiments were performed with enzyme and ligand species in EPPS buffer (pH 8, KOH), 10 mM MgCl2, 100 mM KCl, and 0.1 mM EDTA. Data Analysis—The steady state rate equation describing the interaction of substrate A and allosteric ligand X with the enzyme E is shown [28] in Equation 1. Where v is the observed rate, Vo is the maximal rate in the absence of allosteric ligand, and Wax is the coupling constant reflecting the Vmax influence between A and X. Since ET ϭ [E] ϩ [EA] ϩ [EX] ϩ [AEX], the concentration of the ternary complex can be calculated using the following expression:
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