The measurement of the mass ratio of the fragmentated isomers of both enantiomers of 2b shows that they were formed with inversion of the configuration at the stereogenic C-atom (Figure ). The fact that retention was not observed contradicts hypothesis 1 and is in agreement with hypothesis 3 (Figures ). Interestingly, pro-S decarboxylation was also observed in the wildtype-catalyzed decarboxylation, albeit to a much smaller extent. AMDase produced (R)-2b with 96% ee (Figure ). The mass signal of the (R)-peak showed excess of the signal at m/z 85 over that at 86 (m/z [85]:[86] = 94:6), indicating loss of the labeled carboxylate and hence pro-R decarboxylation. This mode of decarboxylation is also the case for the conversion of aromatic malonates by the wildtype, and is the expected result from these experiments. Surprisingly, however, the smaller peak of the (S)-enantiomer showed an excess of the signal at m/z 86. This indicated conservation of the 13 C-carboxylate and hence pro-S decarboxylation. The (R)selective mutant AMDase IPLL (V43I/A125P/V156L/M159L) also produced unlabeled (R)-2b via pro-R-decarboxylation. With 98% ee, the stereoselectivity was even higher than that of the wildtype (Figure ) and shows that AMDase effectively discriminates between the sterically similar methyl and vinyl substituents. In summary, pro-R decarboxylation by wildtype AMDase formed (R)-2b and pro-S decarboxylation formed (S)-2b. For the wildtype enzyme, both stereochemical pathways proceed with inversion of the absolute configuration at the Ca (Figure ). The mechanism proposed in hypothesis 3 (Figure ) implies that the protonation is part of the rate-limiting step. To investigate this, we measured the D KIE under substrate-saturating conditions. As expected, decarboxylation of 1a showed a D KIE (kH/kD) = 1.05. This low value confirms that decarboxylation is the rate-limiting step for aromatic malonates. In contrast, decarboxylation of 2a had a D KIE (kH/kD) = 1.42, indicating that proton transfer is rate-limiting. Interestingly, the KM-value of 0.11 mM for AMDase ICPLLG towards 2a is 80-fold lower than that for the wildtype enzyme. In contrast, the turnover frequency kcat of 23.2 s -1 is approximately half than that of the wildtype enzyme which has a kcat of 42.8 s -1 . The tight substrate binding by the variant is another striking difference to the wildtype.