been observed in AC crystal structures, 1, 28-30 but, based on NMR studies, the hydrolysis product is suggested to be either the Δ2 or R-Δ1 tautomer. Kinetic measurements suggest that for OXA-48-like β-lactamases, deacylation is the ratelimiting step in carbapenem breakdown. These authors suggested that the impaired imipenemase activity in the ESBL-like OXA-163, compared to OXA-48, is due to a larger active site, which would not constrain the substrate in deacylation-compatible conformations. Molecular dynamics (MD) simulations of the non-covalent complexes of OXA-48 and OXA-163 with meropenem and imipenem suggested some differences between the substrates in mobility. However, the measured KM values for OXA-48 with imipenem and meropenem are very similar (according to one assay, 11 and 13 μM, respectively) , which indicates that there is unlikely to be any significant difference in the stabilities of the respective Michaelis complexes. The difference in the inactivation efficiency of imipenem compared to meropenem is thus primarily related to differences in the rate of the deacylation step, and it is therefore essential to consider this reaction when seeking to understand and explain activity differences. To analyse differences in activity for carbapenems in atomistic detail, we here simulate TI formation in deacylation, i.e. the expected rate-limiting step, of both imipenem and meropenem by OXA-48 using combined quantum mechanics/molecular mechanics (QM/MM) simulations. Our simulations support the hypothesis that the AC state arising from carbabenem acylation is dynamic in nature. Further, we identify conformations of the 6αhydroxyethyl group that allow for efficient deacylation. Additionally, active site hydration around the carboxylated Lys73 is observed to affect the calculated free energy barriers for deacylation, as we previously observed hydrolysis of the expanded-spectrum oxyimino cephalosporin ceftazidime by OXA-48 enzymes. Analysis of the reaction simulations shows that efficient carbapenem breakdown results both from a decrease in hydration around carboxy-Lys73, and from subtle changes in hydrogen bonding between the substrate and the catalytic water molecule. These results provide detailed insight into the causes of differences in enzyme activity against different antibiotics, information potentially useful in understanding and combating antimicrobial resistance.