In a 1 ns trajectory where the dihedral is permanently in Position II (Figure ) a hydrogen bond between the 6α-1R-hydroxylethyl hydroxyl group and ND2 of Asn132 was occupied for 45% of simulation time. Two different conformations of the Asn132 side chain were identified (Figure ), with the highest occupied conformation similar to that observed in the crystal structure of the SFC-1 meropenem acyl-enzyme, and the other similar to that observed in profiles obtained for wild-type TEM-1 when the 6α-1R-hydroxyethyl group is restrained to position II. Free energy surfaces were calculated for the three mutant enzymes to investigate the effect of Asn132 on the energy barrier for meropenem deacylation in SFC-1, TEM-1 and BlaC. The loss of Asn132 increased the barrier for SFC-1 from 10.5 kcal/mol to 12.5 kcal/mol. For the BlaC Gly132Asn mutant, the energy barrier is similar to the wild-type (18.2 kcal/mol vs. 17.9 kcal/mol).