The EVB simulations of the P166A variant of yTIM give 2.7 and 2.2 kcal mol -1 increases in the activation barrier for deprotonation of GAP and DHAP, respectively, that are in good agreement with the 2.6 and 2.3 kcal mol -1 increases in activation barriers determined from the effect of the substitution on kcat for isomerization reactions catalyzed by TbbTIM. We conclude that these simulations model both the effect of the P166A substitution on the structure of the enzyme-ligand complex, and on the activation barrier for TIM-catalyzed deprotonation of enzyme-bound substrate.