Detailed comparison of the simulations revealed two factors that significantly affect the reaction energetics: hydration around Lys73, and the hydrogen bonding pattern between the DW and substrate, specifically the 6α-hydroxyethyl group. Hydration around the general base has been proposed to affect the predicted hydrolysis rates for other β-lactam substrates; 36 here, we show that this is affected by hydration around both Lys73 carboxylate oxygens (not only the oxygen participating in proton transfer). Increased hydration around the non-reactive oxygen (Lys73:OQ2) correlates with higher calculated barriers; in turn, the orientation of Val120 correlates with the number of water molecules near this oxygen. Another aspect influencing deacylation efficiency is the pattern of hydrogen bonds in the active site that involve the DW and the carbapenem 6αhydroxyethyl sidechain. Imipenem shows a preference for a configuration in which the DW donates hydrogen bonds to Lys73 and the 6α-hydroxyethyl hydroxyl group; the free energy barrier is higher when the hydroxyl group instead rotates to donate a hydrogen bond to the DW. This preference is not observed for meropenem: simulations with both hydrogen bond configurations have comparable energy barriers, which are similar to that calculated for imipenem in the less favorable orientation. Therefore, we can conclude that the difference between hydrolytic activities for the two carbapenem substrates stems from subtle differences in the active site hydrogen bonding patterns, which affect the reactivity of the DW. Furthermore, our results indicate that active site hydration is an important determinant of catalysis in OXA-48 enzymes: increasing hydration around the general base impairs carbapenem hydrolysis. Our study highlights the importance of detailed atomistic modelling in addition to experimental research to determine the exact origins of catalytic activity. Simulation protocols such as those employed here can extend information from crystallographic studies to enable investigation of the strength and dynamics of specific active site interactions during the catalytic cycle and directly investigate determinants of activity in situ.