Protein Variants. K12 and E97 Substitutions. The K12M/G15A variant of TIM showed no detectable activity for catalysis of isomerization, and the X-ray crystal structure for this variant revealed that the interaction between the K12 side chain and the substrate phosphodianion is required to observe substrate bound to the crystalline protein. The K12G variant of yTIM is active, but the amino acid substitution results in a 7.8 kcal mol -1 increase in the activation barrier to kcat/KM for TIM-catalyzed isomerization, and much of the lost catalytic activity may be rescued by alkyl ammonium cations. The interaction between GAP and K12G yTIM is too weak to provide clear evidence for saturation. However, the K12G substitution results in a 2.4 kcal mol -1 destabilization of the complex to the intermediate analog phosphoglycolate (PGA) that is consistent with a 2.4 kcal mol -1 destabilization of the Michaelis complex to GAP and a (7.8 -2.4) = 5.4 kcal mol -1 increase in the barrier for conversion of this complex to the isomerization transition state. Our EVB simulations predict increases in the activation barrier to substrate deprotonation (∆∆G ‡ wtàmut) of 3.7 kcal mol -1 for substrate DHAP and 2.5 kcal mol -1 for substrate GAP, compared to wild-type TIM, upon truncation of the K12 side chain to a glycine. By comparison, we obtain similar -3.9 and -2.9 kcal mol -1 electrostatic contributions to the calculated activation free energies for the deprotonation of substrates DHAP and GAP, respectively, by wild-type TIM (Figure ), evaluated by applying the linear response approximation (LRA) to the corresponding EVB trajectories. The calculated 3 to 4 kcal mol -1 effect of the K12G substitution on the activation barrier to substrate deprotonation is smaller than the estimated 5.4