A somewhat unique characteristic of EcPFK is that both of the allosteric ligands, activator MgADP and inhibitor phospho-enol-pyruvate (PEP), bind at the same effector-binding site. This study attempts to understand why two different ligands binding to an identical binding site propagate such markedly different effects. Here we investigated the role that functional groups in PEP play in ligand binding and allosteric propagation via thermodynamic linkage analysis. Previously reported data suggested a potential interaction between PEP and serine 58 in EcPFK contributes substantially to ligand binding and plays a lesser role in allosteric signal propagation. By mutating Ser58 to Ala, Cys, and Asn we have further probed its interaction with PEP. When the Ala, Cys, and Asn mutants were examined, an increase in Kd for PEP of 90x, 30x, and 160x, respectively, that of wild type EcPFK was observed. Each mutant also displays somewhat diminished allosteric inhibition, however allosteric propagation was affected to a lesser degree than binding. In the presence of MgADP the Ala, Cys, and Asn variants exhibit a Kd of 0.25mM, 0.038mM, and 2.8mM, respectively. Comparing the values to wild type EcPFK, with Kd equal to 0.073mM, we see strong changes in Kd while two of the three mutations showed little to no change in allosteric activation. Though these data provide more evidence of a larger role for Ser58 in inhibition relative to activation, no affect on the nature of allosteric phenomena is observed for either MgADP or PEP in the mutants. Also, the examination of inhibitor analogs phosphonoacetic acid, 2-carboxyethylphosphonic acid, and 2-phosphonomethylacrylic acid in the above mutations further strengthens the hypothesis that Ser58 plays a larger role in ligand binding than allosteric coupling. Funding provided by NIH grant GM33216 and the Welch Foundation.