The destabilization of the TI relative to the acyl-enzyme by Asn170 is due to its coordination of the DW and Glu166 in the acyl-enzyme: Asn170 forms two hydrogen bonds with the DW and Glu166. This stabilizes the carboxylate form of Glu166, but lowers the nucleophilicity of the DW similarly to interactions involving the hydroxyethyl group . The increased destabilization of the TI compared to the acyl-enzyme by Asn170 correlates well with the calculated free energy barriers. Asn170 contributes the least to destabilization of the TI in NMC-A, which has both the lowest calculated and experimentally determined free energy barrier to deacylation. All carbapenem-inhibited enzymes (including SFC-1 Cys238Gly) experience greater destabilization of the TI by Asn170 compared to carbapenemase enzymes, except BlaC, indicating the importance of Asn170 destabilization of the TI in inhibiting carbapenemase activity. Furthermore, kinetic and structural studies on GES-type class A beta-lactamases, comparing Asn170 in GES-2 with Gly170 and Ser170 in GES-1 and GES-5 respectively, in the context of otherwise identical active sites, also show the role of Asn170 in coordinating the DW . It has been suggested that the formation of a carbapenem acyl-enzyme complex with GES-1 and -5 causes a displacement of the DW, suggesting a water molecule from bulk solvent would have to enter the active site to facilitate deacylation. In contrast, DW remains in the GES-2 active site due to an additional interaction with the Asn170 side chain, not present in GES-1 or -5. This interaction, however, reduces the nucleophilicity of the DW, attenuating nucleophilic attack of the acyl-enzyme complex. The result is that GES-2 displays poor, but measurable, carbapenemase activity. GES-5, despite requiring a bulk solvent molecule to enter the active site and uptake the position of the displaced DW, does not attenuate the nucleophilic attack because a serine rather than an asparagine is present at position 170. This results in deacylation being more efficient in GES-5 than GES-2 .