The calculated activation free energy barriers (Table ) show good correspondence with apparent barriers derived from published kinetic experiments (kcat values). Table presents the barriers from QM/MM free energy simulations. Our previous results indicated that activation energy barriers for meropenem deacylation lower than around 13 kcal/mol are found for carbapenemases at this level of theory, while barriers above approximately 17 kcal/mol are calculated for carbapenem-inhibited enzymes. These calculated barrier heights are slight underestimates compared to apparent experimental barriers (due to the SCC-DFTB method), but the discrimination between carbapenem-inhibited and carbapenem-hydrolyzing enzymes is well reproduced . This demonstrates that our simulation approach can serve as a starting point for analysis of factors affecting energy barriers in the enzymes considered here. The overall shape of the free energy surfaces for the first step in carbapenem deacylation by the all enzymes, except TEM-1 with meropenem substrate (Figure ), is similar, indicating that the reaction in the different enzymes proceeds in a similar manner. Starting from the acyl-enzyme state (with reaction coordinate values for nucleophilic attack and for proton transfer of 3.5 Å and 0.8 Å, respectively), the reaction follows a minimum energy pathway towards the TI (reaction coordinate values for nucleophilic attack and for proton transfer of 1.5 Å and -0.8 Å, respectively), reaching a transition state that is typically located at values around 1.7 Å and -0.2 Å, except TEM-1/meropenem which is approximately 1.8 Å and -0.6 Å. The shape of those energy surfaces, with the transition state generally in the central part of the plot, indicates that the proton transfer and nucleophilic attack probably happens concertedly, rather than stepwise. (Similar behavior is also observed for the reaction of TEM-1 with the good substrate benzylpenicillin).