There is additional stabilization of the transition state for formation of the enediolate phosphate oxyanion by interactions with other polar side chains and backbone amides at the enzyme active site, most prominently the imidazole side chain of H95, and the amide side chain of N10. These interactions were evaluated as -0.6 (DHAP) and -1.1 kcal mol -1 (GAP) for H95 and -0.2 (DHAP) and -1.1 kcal mol -1 (GAP) for N10, obtained by applying the linear response approximation (LRA) to calculated EVB trajectories for the deprotonation of these substrates by wild-type TIM (Figure and Table in the Supporting Information). By comparison, the H95Q mutation results in a ca. 3 kcal mol -1 increase in the activation barrier for TIM-catalyzed isomerization. In conclusion, the results of experimental and computational studies are converging to provide a clear rationale for the 10 kcal mol -1 stabilization of the transition state for deprotonation of TIM-bound substrate (Table ). The total transition state stabilization represents the combined contributions of many different side chains. The precision in their placement at the TIM active site is the end product of the evolution of a catalytic proteinsubstrate cage that effects a large reduction in the standard-state Gibbs free energy of reaction for proton transfer to the carboxylate side chain of E165, and which is strongly stabilized by interactions with the substrate dianion.