Notably, the barriers are consistently underestimated due to the QM method used (DFTB2), as is generally found for this method for similar reactions. This underestimation likely also causes an underestimation of the stability of the TI compared to the TS (see e.g. the small molecule benchmark calculations the SI section "Benchmarking"), but TI minima were still located in our free energy surfaces (likely due to stabilization by the enzyme environment). As the overall shape of the QM/MM PES is consistent when using DFTB2 or M06-2X/def2-TZVP as the QM method, it is reasonable to expect that the underestimation of TI stability with DFTB2 does not affect trends in reaction barriers (SI section "Benchmarking"). Taking into account an underestimation of ~8 kcal/mol, as indicated by comparison of DFTB2 to SCS-MP2/aug-cc-pVTZ (SI section "Benchmarking"), the lowest barriers are in good agreement with experiment (see further the section "Comparison with experimental data"). Importantly, we expect our protocol for obtaining free energy barriers using semi-empirical QM methods to be a reliable indicator of relative energetic trends between different enzyme active site conformations; we have demonstrated this previously in studies of deacylation of β-lactam acylenzymes for both class A (with meropenem) and D SBLs. As discussed above and in ref. , increased hydration around the proton-accepting Lys73:OQ1 impairs deacylation in ceftazidime hydrolysis. A similar phenomenon was observed for carbapenems, with the additional observation that hydration around the second carboxylate oxygen (Lys73:OQ2) also affects reactivity. In orientation I, the average number of hydrogen bonds Lys73:OQ1 accepts during the reaction is 2.4 (± 0.1 standard deviation, calculated from the US minimum free energy path trajectories), which aligns with OQ1 being hydrogen bonded to two water molecules, and partly to Trp157. The two subpopulations with different deacylation barriers arise from a change in hydration around Lys73:OQ2. For the lower barriers in Figure , the number of hydrogen bonds to OQ2 is 1.3 (± 0.1) and for the higher barriers 2.2 (±0.1) for orientation I. The lowest calculated deacylation barrier, 8.4 kcal/mol, is for imipenem in orientation I with one water molecule hydrogen bonded to OQ2 and two to OQ1 (Figure ). The barrier increases by 2.0 kcal/mol when another solvent molecule donates a hydrogen bond to OQ2. For meropenem, the barrier is raised by 4.1 kcal/mol upon introduction of an additional water molecule close to OQ2.