A complete list of all ionized residues and histidine protonation patterns for each system can be found in the Supporting Information (Table ). Empirical Valance bond (EVB) calculations performed using the Q6 simulation package and the OPLS-AA force field, for consistency with our previous work. Each reaction step/system was simulated for 30 replicas, using an initial 30 ns of equilibration starting from the approximate transition state (λ = 0.5, Figure ). Production simulations were then propagated downhill from the transition state using 51 mapping windows in total, each with a simulation time of 200 ps. Simulation analysis was performed using CPPTRAJ. 3