Figure shows representative structures of the Michaelis complexes, transition states, and enediolate phosphate intermediates for the deprotonation of substrates DHAP and GAP by wildtype yTIM during our EVB simulations. These simulations are performed starting with the highresolution structure of yTIM (PDB ID: 1NEY ), and we therefore use the numbering for yTIM for all amino acid side chains. Table summarizes the activation barriers and reaction free energies for the deprotonation of substrates DHAP and GAP to form enediolate phosphate intermediates (Scheme 1) catalyzed by the wild-type and substituted variants of TIM that were calculated in EVB simulations and comparisons between calculated activation barriers and activation barriers determined by experiment. The bar graphs from Figure compare the calculated and experimental activation barriers. We note that, in several cases, calculated activation barriers are compared to experimental values obtained using enzyme kinetic parameters determined for amino acid substituted variants of TbbTIM. There is strong evidence that the active site catalytic side chains at TIMs from different organisms afford a similar stabilization of the isomerization transition state: (A) The active sites determined by X-ray crystallographic analysis of TIMs with as little as 50% sequence homology are superimposable. (B) The similar kinetic parameters determined for wild-type TIMs from Trypanosomes, yeast, rabbit muscle, and chicken muscle, show that these enzymes provide a similar (± 1 kcal mol -1 ) total stabilization of the rate-determining transition states for isomerization of GAP and DHAP. These results predict similar effects of side chain substitutions on the activation barrier. . ∆G ‡ calc and ∆G ‡ exp denote the calculated and experimental activation free energies, respectively, ∆G 0 calc denotes the calculated reaction free energy, ∆G ‡ TIM -∆G ‡ non denotes the difference between the activation free energy calculated for the TIM-catalyzed reaction and for the background reaction in aqueous solution, ∆G ‡ WT-∆G ‡ mut denotes the difference between the activation free energy calculated for wild-type TIM and for the different TIM variants considered in this work, ∆G ‡ calc-∆G ‡ exp denotes the difference between the calculated and experimental activation free energies for wild-type TIM and the different TIM variants considered in this work, and ∆G 0 TIM -∆G 0 non denotes the difference between the reaction free energy calculated for different TIM variants and for the background reaction in aqueous solution. .