For meropenem, the lowest calculated deacylation barrier is 11.2 kcal/mol with an average of 2.4 (± 0.1) and 1.4 (± 0.0) hydrogen bonds accepted by K73:OQ1 and OQ2, respectively. This barrier is 2.8 kcal/mol higher than the lowest calculated barrier for imipenem, or 2.2 kcal/mol including the free energy penalty (derived from MM MD for imipenem) for orientation I. In contrast to imipenem, the hydroxyl of the 6α-hydroxyethyl moiety in meropenem always rotates during unrestrained US sampling to hydrogen bond configuration (2), donating a hydrogen bond to the DW. This rotation occurs before the TS is reached even when configuration (1) is present in the starting structure. Enforcing the donation of a hydrogen bond from DW to the 6α-hydroxyethyl -OH, i.e. restraining the reaction simulations to configuration (1), affects the location of the TS in a similar manner to that observed with imipenem. TS locations for configurations (1) and ( ) are at -0.2/1.8 Å and -0.5/2.0 Å (proton transfer/nucleophilic attack), respectively. However, changing the hydrogen bonding pattern between configurations has only a minimal effect on the energetics, as the barrier for (1) is 11.9 kcal/mol. Therefore, the decrease in activation energy for configuration (1) vs. (2) does not follow the same trend for meropenem as it does for imipenem. Possible reasons for this may include the presence of a 1β-methyl group in meropenem, as this may hinder the rotation of the 6α-hydroxyethyl group to better optimise further hydrogen bonds between active site residues and water molecules nearby. Such hindrance of 6α-hydroxyethyl rotation may also explain the preference observed for configuration 2 as the DW approaches the acyl carbon. A water molecule lodged between Tyr211 and Thr213 accepts a hydrogen bond from the carbapenem -OH moiety in configuration (1) or donates a hydrogen bond to it in configuration (2) (Figure and Figure ). The 1β-methyl group occupies the space above this water and may therefore induce its displacement or the re-organization of the surrounding water molecules to optimise hydrogen bonds between them, which could subsequently lead to a change from configuration (1) to (2).