Phosphofructokinase from Escherichia coli (EcPFK) is allosterically regulated by MgADP and phospho(enol)pyruvate (PEP). Both molecules compete for binding to the same allosteric site, however, MgADP activates and PEP inhibits the binding of fructose-6-phosphate (F6P) to the active site. The mode by which this enzyme can differentiate between the two ligands and cause the appropriate response is important for the understanding of the basis of allosteric regulation. We studied the interactions between an active site and an allosteric site (heterotropic interactions) within the protein, and found that each of the four unique heterotropic interactions is unique and the magnitudes of the coupling free energies for MgADP activation sum up to 100% that of wildtype EcPFK without homotropic cooperativity in F6P binding. We took on the kinetic and structural characterization of phosphofructokinase from Lactobacillus bulgaricus (LbPFK) to reveal an enzyme that exhibits allosteric properties in spite of previous kinetic studies performed by Le Bras et al. (1991). We have identified residues in EcPFK (Asp59, Gly184 and Asp273), which are important for the allosteric responses to both MgADP and PEP. Interestingly, Lys214 is only important in PEP inhibition and not MgADP activation. We can also differentially disrupt the MgADP heterotropic interactions with the introduction of G184C within the protein. These results suggest that there are different pathways for allosteric communication within the enzyme: different paths for MgADP activation and PEP inhibition, and different paths for each heterotropic interaction with Gly184 being important for the 33Å MgADP heterotropic interaction.