For the lower barriers, Leu158 has not (yet) rotated from the g-to the t rotamer (Figure ), as the starting structures were chosen almost directly after the heating phase. The g-rotamer of Leu158 allows space only for the DW positioned near Lys73:OQ1, which was inserted into the active site in the starting model. Further, only one water molecule is donating a hydrogen bond to OQ2. Upon MD equilibration, Leu158 rotates, allowing for active site hydration to change to two water molecules hydrogen bonding to both carboxylate oxygens each. Subsequently, only the 'high barrier' hydration state is sampled. This explains the large increase in activation free energy when comparing the two hydration substates for orientation III, as two water molecules are located near Lys73, as opposed to only one water molecule close to Lys73:OQ2 (as for orientations I and II). Therefore, our simulations indicate that III is the AC orientation that is the least competent for deacylation for the equilibrated system (in which Leu158 has rotated). Experimentally, this AC orientation is seen in the crystal structure of OXA-48 with hydrolyzed, non-covalently bound imipenem (PDB ID 6PK0) , where the hydroxyethyl hydroxyl donates a hydrogen bond to the newly-formed carboxylate group. In our MM MD simulations of the AC, the exchange between 6αhydroxyethyl dihedral orientations is frequent (indicating a low energy barrier). This is probably true also for the hydrolyzed antibiotic, suggesting that rotation of this moiety can occur postdeacylation.