In our calculations of the AMDase ICPLLG variant, obtained activation energies were closer to the experimental activation energy of 15.7 kcal mol -1 (based on the measured kcat value shown in Table ) for pro-S decarboxylation (19.5 kcal mol -1 ), and a higher activation free energy calculated for pro-R decarboxylation (24.8 kcal mol -1 ) proceeding with inversion of configuration (hypothesis 3). This data is shown in Figure , and the corresponding energies of each individual reacting state as well as key distances at each stationary point are shown in Tables . Figure 5: Graphical analysis of the QM/MM well-tempered metadynamics simulations performed to simulate the carboxylate cleavage and protonation of methyl vinyl malonate by the AMDase ICPLLG mutant, performed at the ωB97X-D3/6-31+G(d) level of theory with 80 ps of sampling time. The data shown here correspond to; A.-B. free energy landscapes for the pro-S (A) and pro-R (B) decarboxylation reactions, proceeding with inversion of configuration, as well as C.-D. The corresponding time evolution of key reacting distances across the reaction path for each mechanism (with black, the hydrogen bond between the cleaved CO2 and G190). The corresponding energy data is shown in Table , and the distance values are summarized in Tables and, for pro-S and pro-R decarboxylation, respectively. E. Atomistic depiction of representative structures of the reactant state (RS), decarboxylation transition state (TS1), intermediate state (IS), proton transfer transition state (TS2) and product state (PS) of the reaction with 2-methyl-2-vinyl malonate (2a) substrate in the AMDase ICPLLG mutant with pro-S inversion mechanism. The substrate and the catalytic cysteine side chain (QM region) carbon atoms are colored purple, the hydrogen bond donor G190 carbons are colored green. The other atoms are colored according to standard colors available in Pymol.