Based on these parameters, we obtain an activation free energy of 17.5 ± 0.6 kcal mol -1 for the analogous reaction catalyzed by wild-type HisA, which is only 1.1 kcal mol -1 higher than the experimental value (derived from kcat) of 16.4 kcal mol -1 . To further validate our PriA/HisA-ProFAR results, we performed single amino acid substitutions in each enzyme (R19A and D130A