Failure of a two-state model to describe the influence of phospho(enol)pyruvate on phosphofructokinase from Escherichia coli

A linked-function analysis is presented of the influence of the inhibitor phospho(enol)pyruvate (PEP) on the binding of fructose 6-phosphate (Fru-6-P) and MgATP to phosphofructokinase (PFK) from Escherichia coli. The results of this analysis indicate that the previously described inhibition of Fru-6-P binding by MgATP [Johnson, J. L., & Reinhart, G. D. (1992) Biochemistry 31, 11510-11518] is almost completely independent of the inhibition by PEP. Moreover, with or without the presence of MgATP, the inhibition by PEP does not conform to the behavior expected if PEP and Fru-6-P bind exclusively to different enzyme forms since the formation of a ternary complex with both PEP and Fru-6-P bound is clearly evident at high concentrations of Fru-6-P and PEP. van't Hoff analyses of the coupling interactions between PEP and Fru-6-P in the presence and absence of MgATP indicate that these couplings are driven by enthalpy. However, the influence that PEP has on MgATP binding is small and changes from being activating to being inhibitory at temperatures above 40 degrees C, revealing the importance of a compensating entropy component to the coupling interactions. The four functionally defined enzyme forms that contribute to the coupling between Fru-6-P and PEP were evaluated structurally using the fluorescence properties of the single intrinsic tryptophan as a probe. The steady-state and dynamic fluorescence emission and polarization properties of the tryptophan, as well as its susceptibility to I- quenching, indicate that the flexibility of PFK in the vicinity of the tryptophan is perturbed by the binding of ligands. The properties of free PFK do not lie between those established by the binding of Fru-6-P and PEP individually, indicating that it is structurally distinct. The properties of the ternary complex lie between those of the singly ligated forms. Though an equilibrium mixture of two conformations of ternary complex cannot therefore be formally ruled out, no evidence obtained to date requires the presumption of this mechanistic complication.