Following our prior success in using the empirical valence bond (EVB) approach to study a wide range of analogous ring opening reactions, such as lactone and epoxide hydrolysis, we extended this approach to study the enzyme-catalyzed opening of the ribose ring of substrates ProFAR and PRA (Figure ), as catalyzed by wild-type and variant forms of HisA, PriA, and TrpF. Our focus for our EVB simulations was specifically on the ribose ring-opening reaction (the first step of the mechanism shown in Figure ), as motivated in the Results and Discussion, and described using the valence bond states shown in Figure . Simulations were performed on wild-type SeHisA, MtPriA and TmTrpF as well as selected variants, as described in the Supporting Information. All simulations were performed using the Q6 simulation package, using the OPLA-AA force field. All EVB parameters necessary to reproduce our work, as well as a detailed description of the computational methodology and subsequent simulation analysis can be found in the Supporting Information, with the full parameters used in our simulations updated to Zenodo (DOI: 10.5281/zenodo.5893598). Each system was simulated using 30 individual replicas, with each replica first equilibrated for 20 ns and the endpoint of that equilibration being used as the starting point for propagating an EVB trajectory. Each EVB free energy perturbation/umbrella sampling (EVB-FEP/US)