The experimental and computational results show that the contribution of the anchoring K12•E97 ion pair to the enzymatic rate acceleration is < 1 kcal mol -1 . EVB simulations likewise show that the E97A substitution of K12G TIM to give the double K12G/E97A substitutions results in a ≤ 0.3 kcal mol -1 increase in the calculated activation barriers for the K12G yTIMcatalyzed deprotonation of DHAP and GAP, and that the effect of the K12G substitution on activation barrier for catalysis by the E97A variant lies within 0.3 kcal mol -1 of the effect of the K12G substitution on the activation barrier for catalysis by wild-type yTIM. We conclude that the role of the E97 side chain in preorganization of the K12 side chain at a K12•E97 salt bridge does not provide for strong stabilization of the isomerization transition state. This is consistent with the conclusion that the evolutionary pressure to optimize the activity of TIM, which plays a critical metabolic role in cellular energy production by glycolysis, is so strong that the E97 side chain is strictly conserved in order to obtain a ca. 1 kcal mol -1 stabilization of the rate determining transition state. The E97 carboxylate anion is hydrogen bonded to the T75 side chain at the second subunit of TIM. The placement of this side chain in the middle of a network of hydrogen bonds that spans the K12 and T75 side chains at neighboring protein subunits implies an additional role for E97 in maintaining the dimeric structure for TIM, which merits investigation.