The E97A, E97D and E97Q substitutions of yTIM each result in ≤0.7 kcal mol -1 changes in the calculated activation barriers for wild-type TIM-catalyzed deprotonation of DHAP and GAP (Table ). These changes are within the uncertainty of these simulations and represent increases in the activation barrier, except for the 0.7 kcal mol -1 transition state stabilization calculated for the E97D variant of yTIM. There is good agreement between the calculated effect of these three substitutions on the activation barrier for catalysis by wild-type yTIM and the experimental activation barrier (where available) for catalysis by TbbTIM (Figure ). The LRA simulations (Figure ) are consistent with a ~1 kcal mol -1 destabilizing contribution to the activation barrier for TIM-catalyzed deprotonation of DHAP and GAP from interactions between the side chain anion of E97 and the distant anionic transition state.