Focusing on kcat, which reflects the rate-determining step in turnover, the variants show alterations in the pH optimum, in their pH-independent limiting kcat values, and the kinetic pKa values obtained from fits to Eq. 1 (Table ). The kinetic pKa values for the aspartic acid for the variants are each ~1 unit higher than the WT, reflected in their broader pH-rate profiles. The native SHP-1 has a kinetic pKa of the aspartic acid of 5.6, while in the variants, it is increased to 6.9, 6.7, and 6.5 for H422Q, E427A, and S418A, respectively. Though the variants and WT have similar activities at their optimal pH, all of the variants have faster turnover at pH > 6. In contrast, the pH-independent limiting kcat values show minimal difference between the different enzyme variants, an observation also supported by empirical valence bond simulations (Table ). For comparison, Table lists the previously reported kcat values obtained in acetate buffer at pH 5.0 with added 40% glycerol. The pKa of carboxylic acid moves higher in nonaqueous solvents and in cosolvent mixtures. The pKa of acetic acid in glycerol/water mixtures has not been reported to our knowledge, but is increased by a full unit in 50% ethanol. It is possible that the aqueous glycerol conditions were at an effective pH of 6 or above, in the range where our aqueous solution kcat values for the variants are higher than WT (Figure a The pKa1 value arises from the cysteine nucleophile and pKa2 from the aspartic acid. Shown here for comparison are reported kcat values (s -1 ), in 40% glycerol solution, pH 5. 10, 25 b Data from ref. . c Data from ref. . d Calculated activation free energies, in kcal mol -1 , presented as average values and standard error of the mean across 30 independent EVB trajectories propagated for each system, performed as described in the Materials and Methods.