The DHFR system. The DHFR model used the reactant state from Luk et al. as the starting structure. The QM region, with a net charge of +1, was constituted by 69 atoms, including cofactor's nicotinamide and ribose rings; substrate's pteridine ring and the N-methylene-substituted p-aminobenzoyl group; and the two hydrogens as link-atoms saturating the valences at the QM-MM boundaries (see DHFR scheme in Figure ). This structure underwent MM-level minimization using the steepest descent algorithm to avoid clashes in both QM and MM zones. A subsequent 200 ps of QM/MM NVT equilibration was performed at 300 K with a 1 fs time step. Starting from the relaxed reactant structure, we identified the product state through a scan calculation, employing the hydride transfer antisymmetric distance as the reaction coordinate. The equilibrated product state underwent the same simulation conditions as the reactant for 200 ps, with the final 40 ps used to compute averaged collective variable (CV) values for ASM calculations. PBC were used for both QM and MM regions, combined with 8Å cutoffs in direct space.