The crystal structures of PchB (PDB entry 3REM ) and BsCM (PDB entry 3ZO8 15 ) were used as starting models for the EVB simulations. For PchB the chorismate was manually built in to the active site by modifying the existing salicylate and pyruvate present in the active site of 3REM. The chorismate configuration for BsCM was obtained by manually modifying the transition state inhibitor present in the Arg90Cit BsCM with PDB entry 3ZP4 and then superimposed to the wild type BsCM (PDB entry 3ZO8 ). Topologies for all simulations were prepared with Q 29 and Qgui 30 where parameters were assigned in accordance with the OPLS-AA/M force field. The enzyme structures were solvated in a spherical droplet of TIP3P water molecules with a simulation sphere radius of 36 Å, centered at the protein center of mass and encompassing the entire protein. A 25 Å radius was used for the chorismate to prephenate reference reaction in water. The systems were equilibrated prior to the MD/EVB simulations by gradually increasing the temperature from 1 K to the final temperature (283 -308 K) through a stepwise scheme (6 steps). The first 5 steps were simulated for 10 ps whereas the final equilibration step was simulated for 100 ps with a time step of 1 fs. The temperature was controlled by coupling the system to an external bath with a relaxation time of 10 fs for the first 5 steps and 100 fs for the final equilibration step and the productive phase. Long-range electrostatics were treated using the multipole expansion method (LRF) and a direct 10 Å cutoff for nonbonded interactions. The reacting fragments were however allowed to interact with the entire system (no cutoff). Bonds and angles of solvent molecules were constrained with the SHAKE algorithm. The productive simulations comprised 51 discrete free energy perturbation (FEP) steps, where each step was simulated for 100 ps, resulting in 5100 ps for each run.