The notion that substrate affinity is modified via interaction of effector ligands is a fundamental characteristic of allostery. In order to further understand the complexities of this phenomenon, this study investigates the role that functional groups in phospho-enol-pyruvate PEP play in ligand binding and allosteric propagation. Thermodynamic linkage analysis enables the nature and magnitude of allostery to be determined, allowing allosteric action to be distinguished from allosteric ligand binding affinity. By using this type of analysis to compare the inhibition caused by PEP to the inhibition imposed by PEP analogs upon binding to E. coli PFK (EcPFK), the importance of specific functional groups of PEP to ligand binding and/or allostery can be ascertained. EcPFK displays a much weaker binding affinity for the PEP analog phosphoglycolate (PG) compared to PEP. However, PG is still able to inhibit EcPFK to an extent comparable to that of PEP. This finding suggests that the methylene group that is absent in PG plays a larger role in ligand binding than in allosteric propagation. The chemical structure of phosphonoacetic acid (PA) is very similar to that of PG except for the deletion of the oxygen that bridges the phosphate group to the rest of the molecule. When the inhibition of EcPFK by PA is examined, a binding affinity comparable to PG is observed but a substantial loss in allostery is also observed. These data suggest that the bridging oxygen in PEP contributes substantially to the propagation of allosteric signal. Funding is provided by NIH grant GM33261 and Welch Foundation grant A1543.