The P166A and I170A substitutions result in 0.7 and 2.3 unit decreases, respectively, in the pKa for the carboxylic acid side chain of E165 at the complex between TIM and the enediolate phosphate analog PGA. These decreases in pKa are proposed to result from change in the solvation of the carboxylate discussed above, that favors the calculated increases in ∆G 0 for proton transfer at these TIM variants (Figure ).