Prompted by the confirmation that AMDase ICPLLG decarboxylates 2a via alternative binding modes (one of them proceeding via the loss of the pro-S carboxylate), we hypothesized that these binding modes should enable AMDase to accept substrates with an increased size of the small substituent. AMDase wildtype is restricted regarding the smaller substituent, and 2ethyl-2-phenyl malonic acid was not accepted as substrate. Chemical decarboxylation of 3a at elevated temperature or under weakly acidic conditions, respectively, led to the migration of the double bond and the formation of the product isomer 2-ethyl-2-butenoic acid (3c) (Figure ). As expected, the wild type enzyme did not show any activity towards 3a. We were delighted to find that AMDase ICPLLG completely converted 3a to pure (S)-3b in outstanding optical purity (>99.5% ee). The capacity of the AMDase variant to discriminate between the sterically highly similar ethyl and vinyl substituents is a striking example of the exceptional enantioselectivity of biocatalysts. Furthermore, the reaction proceeded also in a highly regioselective manner without measurable double bond isomerization. , and the distance values are summarized in Tables and, for pro-S and pro-R decarboxylation, respectively. Determination of the kinetic parameters of 3a revealed a much lower kcat of 0.005 ± 0.0003 s - 1 . Interestingly, the measured KM value of 0.16 mM was very similar to those of AMDase wildtype and AMDase ICPLLG towards 2a. For both substrates, the KM values of AMDase ICPLLG are lower than those of the wildtype. Interestingly, the KM value of 0.11 mM of AMDase ICPLLG towards the sterically much less demanding 2a is very low compared to the KM values of AMDase and its variants towards aromatic malonates, which are usually in the millimolar range. The similar KM values of the mutant towards 2a and 3a raise the question whether the low turnover frequency is caused by a potentially lower reactivity of 3a or by a steric effect. QM/MM well-tempered metadynamics simulations with the ωB97X-D3 functional and the 6-31+G (d) basis set, and the same QM region as with 2a, predict the preference of the pro-S decarboxylation of 3a by the ICPLLG mutant, with a calculated transition state energy of 22.7 kcal mol -1 (Table ), close to the experimentally derived activation energy of 20.7 kcal mol - 1 .