In the case of MtPriA we modeled three single amino acid substitutions (R19A, D130A and R143A) and extended our EVB simulations to model the effect of these substitutions (Table , Figure ), in order to specifically capture the impact of the loss of electrostatic contribution of each truncated side chain on the activation free energy. In the case of the R19A variants, we obtain excellent agreement with the experimental value. However, in the case of the D130A variant, our model significantly under-estimates the activation free energy difference compared to experiment, again suggesting that the experimental effect is rather related to a change in substrate positioning or loop dynamics, that is not captured in our EVB simulations. In the case of the R143A variant, we obtain an activation free energy 1.9 kcal mol -1 lower than the wild-type enzyme. We note that, experimentally, this substitution has been shown to significantly impair the isomerization activity of MtPriA towards PRA (kcat/KM reduced from 1.7 x 10 5 M -1 s -1 to 6.0 x 10 3 M -1 s -1 on introduction of this substitution ). However, it is unclear if this is an effect on kcat, KM or both, and it is plausible that the loss of activity is due to structural effects that prohibit productive substrate binding, that are not captured in our simulations. This latter issue would be similar to our observations from a recent study of an analogous system activated by a ligand-gated conformational change, glycerol-3-phosphate dehydrogenase (GPDH). In this system a substantial loss of activity upon truncation of a key catalytic arginine to alanine could only be explained by structural rearrangements (predominantly blocking of the closure of a key catalytic loop), that were observed upon crystallization of this variant. In contrast, this loss of activity could not be captured simply by performing a truncation of this side chain on the wild-type enzyme and considering only electrostatic effects which only accounted for a smaller part of the observed change in activity. and). Due to the lack of relevant crystal structures, we used structures with loop 1 in an open conformation extracted from our conventional MD simulations as starting points for the EVB simulations. As can be seen from these data, for the catalyzed isomerization of PRA, when the reaction was modelled with loop 1 in an open conformation, we obtain much higher activation free energies for ring-opening than when modeling the reaction from a loop-closed conformation, due to a combination of the loss of key interactions between loop 1 and the substrate, and also extra solvent-exposure of the active site when this loop opens up. However, we observe no impact on the activation free energy when modelling the catalyzed isomerization of ProFAR, staring with loop 1 in open conformation.