As presented above, orientation I of the 6α-hydroxyethyl moiety is calculated to give the overall lowest deacylation free energy barriers for both carbapenems. The combined FESs for the hydration state with lower free energy barriers are presented in Figure for all three substrate orientations. In this section, we focus further on orientation I and the 'reactive' active site configuration in which only one water molecule is hydrogen bonded to Lys73:OQ1, and two to Lys73:OQ2 (unless otherwise stated). For this AC conformation, two different hydrogen bonding arrangements in the active site are possible: the DW can donate a hydrogen bond to the 6αhydroxyethyl hydroxyl group (named configuration 1), or the hydroxyl group can donate a hydrogen bond to the DW (configuration 2), see Figure . In MM MD, configuration (1) is sampled for 87% and 86% of simulation time for imipenem and meropenem, respectively. In addition to donating a hydrogen bond to the DW as in ( ), the 6α-hydroxyethyl hydroxyl group can also donate a hydrogen bond directly to Lys73:OQ1 if the DW is displaced. This orientation of the carbapenem 6α-hydroxyethyl group may be the relevant one for β-lactone formation, which has been characterized as a side product for OXA-48-catalysed carbapenem turnover, particularly of 1βmethyl carbapenems (such as meropenem). The β-lactone product has been proposed to form via intramolecular cyclisation, where the hydroxyl group acts as a nucleophile and donates a proton to Lys73. If the reaction occurs without a bridging water molecule, i.e. by a direct proton transfer between -OH and Lys73, lactonization is most likely lower in energy in orientation I than in III, based on the trends observed for deacylation energetics.