We note that the calculated activation free energies for this step are not trivial to directly compare with the experimental turnover numbers: no kinetic data exists on the rates of the individual chemical steps. Furthermore, the observed kcat values for these systems are extremely low -on the order of 1 s -1 (or lower) for both the HisA and TrpF reactions catalyzed by HisA and its variants. However, E. coli TrpF has a kcat value of 30-40 s -1 , with the rate-limiting step being a spontaneous keto-enol tautomerization step that occurs off the enzyme, after the ring-opening step. When also taking into account the potential involvement of loop dynamics in determining the turnover rates, as is the case in other enzymes with catalytically important conformational changes such as protein tyrosine phosphatases, this means that the experimental turnover numbers for the isomerization of ProFAR and PRA by the enzymes of interest do not correspond to a chemical step occurring in the enzyme active site. However, as the Amadori reaction that occurs between the enzyme-catalyzed ring-opening reaction and the non-enzymatic tautomerization is likely to be fast (even the uncatalyzed reaction occurs spontaneously at 25 °C85 ), the ring-opening reaction is likely the slowest enzyme-catalyzed chemical step in the catalytic cycle. This is supported by QM/MM studies of the mechanism of the HisA-catalyzed reaction, although this work does not take into account that chemistry is not rate-limiting here. However, even if the experimentally measured turnover numbers (kcat) do not directly correspond to this step, they do produce a lower limit for the rate of this step (and thus an upper limit for the corresponding activation free energy for the rate-limiting step of the enzymatic reaction). Thus, comparing the calculated activation free energies for the ring-opening in different variants, and in different conformational states of loop 1, can still provide insight into the impact of loop dynamics and key amino acid substitutions on the rate of the slowest enzyme-catalyzed step.