The glycolytic pathway is the most basic energy production mechanism common to all living things. A drug designed by targeting a bacterial glycolytic protein does not interact with the protein in humans, even if the same protein and the same mechanism are found in both organisms. Since catalytic regions are vital to the function of the protein, these regions are more conserved during evolution, while allosteric regions are more open to structural or sequential changes. The Constraint Molecular Dynamics simulation method has been used to examine the effects of molecules that provide allosteric inhibition of the glycolytic enzymes. Unlike the other MD simulations, this method is not require addition of a parameterized ligand in to the system. Instead, residues known to interact with the ligand are restricted in the specified region, limiting the movement of the residues as if there was a ligand there. In this study, we clarify how glycolytic enzymes are allosterically inhibited using various analysis methods.