We present the results of the most extensive computational study of the effect of substitution of key active sites residues on the activity of triosephosphate isomerase reported to date. These data provide biochemical insight into the mode of enzyme action, while probing the limits of the EVB approach in modeling this deceptively simple proton transfer reaction. The EVB simulations reproduce both the changes in enzyme structure determined for the P166A, I170A, and L230A TIM variants, and the changes in the experimental activation free energies for the broad range of protein variants reported in Table . In addition, the simulations provide thermodynamic barriers to substrate deprotonation, that were used in the construction of linear free energy relationships with excellent linear correlations between the kinetic and thermodynamic barriers for the deprotonation of substrates DHAP and GAP over a 10.4 and 14.9 kcal mol -1 range in activation and reaction free energies respectively. This extended LFER (Figure ) shows that the evolution of the remarkable catalytic proficiency of TIM was driven by the selection of caged enzyme-substrate active site structures that minimize the thermodynamic barrier for the highly unfavourable substrate deprotonation.