However, these conclusions are based on a single 10 ns MD simulation, which likely gives insufficient time to sample all available substrate orientations. Based on our MM MD simulations, as well as the calculated free energy barriers, orientation II is less likely to contribute to efficient deacylation of the carbapenem ACs. This is due both to an increase in energy required for deacylation, as well as to a lack of sampling of active site configurations that would be suitable for the AC carbonyl to undergo nucleophilic attack by an incoming water molecule.