and BlaC (Asn132 substituted by Gly). Umbrella sampling QM/MM MD simulations of reaction were performed as above, but with a restraint on the hydroxyethyl group dihedral to maintain it in position I, II or III. Results for these simulations are also presented in Table . For the TEM-1/meropenem complex, Position I dominated both in QM/MM MD simulations of acyl-enzyme and of the reaction. The barrier for this system is not affected by applying a restraint to keep the dihedral in position I (17.3 kcal/mol compared to an average of 17.1 kcal/mol without restraint), this is as expected because the dihedral adopts this conformation without restraints. For the carbapenemase SFC-1 (and indeed also for KPC-2 and NMC-A), both positions II and III are observed (Figure ). When restraints are applied to the position, a slightly higher barrier is observed with the dihedral in position III, whereas a decrease in barrier by 3 kcal/mol (compared to an unrestrained simulation) is observed with the group in position II. Reaction simulations for TEM-1 with a restraint holding the group in Position II were also performed, and showed an increased activation barrier (by 2 kcal/mol). For BlaC, reaction simulations without dihedral restraints and with the dihedral restrained to position II gave similar activation free energies (Table ). This indicates that, by itself, maintaining the hydroxyethyl group in position II is not sufficient to confer carbapenemase activity. The main structural difference between benzylpenicillin and meropenem that is likely to affect mechanistically important interactions within the -lactamase active site is the presence of the C6 benzamido and α-1R-hydroxyethyl groups, respectively. Although the benzamido group of benzylpenicillin is much larger than the 6α-1R-hydroxyethyl group of meropenem, it does not form any direct hydrogen bonds with the DW or Glu166 and is oriented away from the immediate reaction center. In contrast, the hydroxyl of the 6α-1R-hydroxyethyl group is able to form a direct hydrogen bond with the deacylating water (Figure ), which has been suggested to influence its nucleophilic properties . To further investigate the possible importance of interactions involving the carbapenem 6α-1R-hydroxyethyl group, the behavior of the deacylating water was compared in simulations of the TEM-1 benzypenicilin and meropenem acyl-enzymes. Hydrogen hydroxyethyl group forms a direct hydrogen bond with the deacylating water (DW). This additional interaction displaces the DW, increasing the distance between the DW and CC (Figure ) of meropenem compared to the benzylpenicillin acyl-enzyme (C), values obtained from three independent 1 ns QM/MM MD simulations of each system