For imipenem deacylation, both configurations (1) and ( ) were observed in umbrella sampling. The lowest free energy barrier of 8.4 kcal/mol was calculated for configuration (1), and the barrier was increased by 2.0 kcal/mol for configuration (2). In addition to raising the free energy barriers, changing from (1) to (2) shifts the location of the transition state on the FES. For (1), the TS is located approximately at values -0.1 Å and 1.7 Å for the proton transfer and nucleophilic attack reaction coordinates, respectively (Figure , left). However, for (2), the TS location on the FES shifts to around -0.5 Å and 2.0 Å Figure ). With active site configuration (2), the proton transfer has progressed further at the TS, whereas the approach of the DW oxygen to the acyl carbon is less advanced. This is potentially due to the additional hydrogen bond from the 6α-hydroxyethyl moiety hydroxyl decreasing the nucleophilicity of the DW, requiring the proton transfer reaction to have progressed further from the starting structure in the TS. Notably, a similar shift in the TS position on the FES is observed also in orientation III, where a water molecule is donating a hydrogen bond to the DW instead of the 6α-hydroxyethyl group (Figure ). Mulliken charge analysis of the key QM atoms does not reveal many significant differences for the calculated charges along the reaction when comparing US calculations with either configuration (1) or (2) (Tables ). The main difference is observed at the TS, where for Lys73:OQ1 the charge is more positive and for DW:O the charge is more negative for configuration (2), as expected by the shift in the TS location towards the TI.