Most of the variants in the OXA-48 family are carbapenemases, with elevated imipenem hydrolysis rates when compared against other carbapenems. For OXA-48, experimental measurements of kcat values for imipenem hydrolysis vary between 1.5 and 22.5 s -1 , which can be converted to free energy barriers for activation (Δ ‡ G) of 15.7 to 17.3 kcal/mol, using the Eyring equation. For meropenem, the measured kcat values range between 0.07 -0.16 s -1 , which converts to barriers of 18.7-19.2 kcal/mol. Using these figures as experimental estimates of free energies of activation, the difference (ΔΔ ‡ G) between imipenem and meropenem hydrolysis is between 1.4-3.5 kcal/mol, which is approximately the same magnitude as the strength of a single hydrogen bond (1- 3 kcal/mol). Hence, structural factors contributing to more efficient breakdown of imipenem, compared to 1β-methyl carbapenems, are most likely to be subtle. Our QM/MM simulations suggest that orientation I of the 6α-hydroxyethyl group is the most likely AC orientation to undergo deacylation, when this exists in a state with decreased hydration around Lys73:OQ2 (i.e., with only one water molecule donating a hydrogen bond to this carboxylate oxygen). When comparing the lowest free energy barriers calculated in orientation I for imipenem and meropenem (Figure ), the difference (ΔΔ ‡ G) for the two substrates is 2.8 kcal/mol; including the free energy penalty for the imipenem 6α-hydroxyethyl moiety adopting orientation I (0.6 kcal/mol, as determined from our MM MD simulations), the obtained ΔΔ ‡ G value drops to 2.2 kcal/mol. This is in excellent agreement with the experimentally determined range of ΔΔ ‡ G values. This strongly supports our assumption that TI formation is the rate-limiting process for carbapenem hydrolysis by OXA-48, consistent with similar findings for ceftazidime breakdown by OXA-48-like enzymes and carbapenem breakdown by a range of class A serine β-lactamases. The agreement further implies that the difference between imipenem and meropenem deacylation in OXA-48 may indeed be caused by the subtle difference in the preferred hydrogen bonding patterns involving the DW and the 6α-hydroxyethyl sidechain reported here. In turn, the presence of the meropenem 1β-methyl group apparently contributes to this difference by influencing both the orientation of the 6αhydroxyethyl group and the organization of water molecules in the near vicinity. (We further note that the underestimation of the absolute barriers can be fully accounted for by comparison of DFTB2 to higher level QM calculations, which indicates DFTB2 underestimates barriers by ~6.3-8 kcal/mol, see Table & Figure . Thus, combined with the free energy penalty of 0.6 kcal/mol noted above, the corrected lowest barriers would be 15.3-17.0 and 17.5-19.2 kcal/mol for imipenem and meropenem, respectively.) Based on our MD simulations, the carbapenem tail groups are highly flexible and are thus unlikely to directly affect deacylation efficiency. Differences in kcat (reflecting the rate-limiting deacylation step) for carbapenems might therefore be explained similarly to our findings here, with differences largely caused by the presence or absence of the 1 β-methyl group. This is consistent with experimental data for OXA-48, which show higher kcat values for imipenem and panipenem vs. 1β-methyl containing carbapenems. Overall, our analysis of the effects of active site conformations on carbapenem hydrolysis activity highlights the importance of controlling water access to the active site. On the one hand, it is crucial for the enzyme active site to support the binding of the deacylating water (through the aforementioned water channel). On the other hand, partial desolvation of the catalytic base (carboxylated Lys73) is required for efficient reaction. Such intricate control of active site solvation is a common feature of enzyme activity. For example, in ketosteroid isomerase, additional water molecules hydrogen bonding to the catalytic aspartate raise the barrier of reaction significantly. Notably, this increased solvation occurs through water molecules hydrogen bonding to the carboxylate oxygen that is not receiving the proton, similar to what is observed here (difference between high and low barriers in Figure ), but different from what we observed for ceftazidime hydrolysis. Such additional hydrogen bonding will decrease the pKa of the catalytic carboxylate base, weakening its proton affinity and thereby leading to higher barriers for the reaction. To avoid or limit the occurrence of additional hydrogen bonding to catalytic bases, enzymes have evolved active site architectures that can promote desolvation to increase carboxylate reactivity. Such desolvation can for example be achieved by loop closure (as in triosephosphate isomerase and dihydrofolate reductase) or closure of the substrate binding cleft (as in ketosteroid synthase).