In the case of the substrate PRA, we again used the reaction catalyzed by wild-type MtPriA as our EVB reference state, this time with the loop 5 in its pro-PRA conformation. We note that as shown in Table , wild-type SeHisA does not show TrpF activity, whereas variants in which loop 1 is extended through dup13-15 do. In the SeHisA(dup13-15) variant, we obtained an activation free energy of 19.9 ± 1.2 kcal mol -1 , which is within 1.3 kcal mol -1 of the experimental value (derived from kcat) of 18.6 kcal mol -1 (noting again that the rate-limiting step for the enzymecatalyzed reaction occurs off the enzyme, 86 so this value is only a proxy for the barrier for the ringopening). We then extended our EVB calculations to model PRA ring-opening as catalyzed by a set of variants of MtPriA and SeHisA(dup13-15) (Figure and Table ). In the case of the SeHisA(dup13-15) variants, all the variants yielded results within reasonable agreement (~1 kcal mol -1 ) with experimental values. We note as an aside that we also performed simulations on wildtype HisA for PRA substrate (which is not active toward this substrate) and obtained an activation free energy of 19.5 ± 0.6 kcal mol -1 , very similar to the one obtained for SeHisA(dup13- 15), suggesting that in theory the wild-type enzyme could catalyze this reaction if all loops are in the correct conformation and the substrate is optimally positioned, and that the experimental lack of activity is not due to a high barrier to the chemical reaction catalyzed by this enzyme. Tying in with this, as described in the Supporting Information, our PRA simulations are initated from an idealized position of this substrate in the active site, based on overlay with the position of the larger substrate ProFAR. However, the stability of this ProFAR conformation in the active site is facilitated by interaction with the gripper loop 3, whereas PRA lacks the distal phosphodianion group of ProFAR and is thus not able to make this interaction (Figure ). This suggests that if PRA could be gripped properly (and thus optimally aligned), turnover could in turn happen.