The side chains examined in our studies make two distinct contributions to the thermodynamic/kinetic activation of TIM for deprotonation of bound substrate. First, the transition state for proton transfer is stabilized by 3.7 and 2.5 kcal mol -1 for deprotonation of DHAP and GAP, respectively, through interactions with the cationic side chain of K12. These cations provide no significant stabilization of the transition state for proton transfer in water at pH ≥ 7, where there is no significant catalysis of deprotonation of GAP or DHAP by tertiary ammonium cations. Second, the hydrophobic side chains of P166, I170 and L230 occupy positions that minimize the thermodynamic and kinetic barriers to substrate deprotonation. There is good experimental evidence, described above, that the effect on the reaction driving force is due mainly to the enhancement of the basicity of the carboxylate side chain of E165 at TIM compared to aqueous solution. The effects of the P166A, I170A and L230A substitutions on the activation barriers for deprotonation sum to 3.6 and 6.1 kcal mol -1 , respectively, for deprotonation of DHAP and GAP. This serves as an upper limit for the contribution of these side chains to catalysis in the event that the effects of these mutations are additive.