Computational methods and details of the system setup are described in detail in the Supporting Information (SI). To summarise, models of OXA-48 with imipenem and meropenem were prepared based on corresponding acylenzyme (AC) crystal structures (PDB IDs 6P97 1 and 6P98 1 for imipenem and meropenem, respectively). The ff14SB parameter set was used for the protein, parameters and partial charges for non-standard residues (acylated carbapenems and carboxylated lysine) were derived with the R.E.D. Server. Both systems were energy minimised, heated from 50 K to 300 K (in 20 ps), and their dynamics in the AC state were simulated for 200 ns using Langevin dynamics (collision frequency 0.2 ps - 1 ) with a 2 fs timestep. Five independent simulations for each AC system were run. All bonds involving hydrogens were restrained using the SHAKE algorithm. Starting structures for QM/MM 39 modelling were chosen from MD simulations based on visual inspection of the active site hydration pattern and the 6αhydroxyethyl orientation; this orientation was kept from changing during subsequent QM/MM US MD by applying a weak dihedral restraint (except in the case of orientation I). Free energy barriers for the first (rate-limiting) step of deacylation for the different active site conformations were determined from three separate QM/MM umbrella sampling (US) calculations for each conformation. Two reaction coordinates were employed in US, one for the nucleophilic attack and one for the proton transfer, as in previous simulations of deacylation in serine β-lactamases. Sampling time in each window was 2 ps, and DFTB2 (SCC-DFTB) was used as the QM method for regions consisting of 43 and 46 atoms (including link atoms) for imipenem and meropenem, respectively (Figure ). Free energy surfaces (FESs) were constructed from 399 individual US windows. The weighted histogram analysis method (WHAM) was used to construct the free energy surfaces, and the minimum energy paths were analysed using the Minimum Energy Path Surface Analysis (MEPSA) program . All simulations and trajectory analyses were done using the Amber18 software package (pmemd.cuda for MM MD, and sander for QM/MM calculations).