(3) The D KIE of (kH/kD) = 1.42 indicates rate-limiting protonation. Our findings rule out hypotheses 1 and 2. The observed cleavage of the pro-S-carboxylate contradicts hypothesis 2, while the inversion of stereoconfiguration after pro-R decarboxylation in the mutant disproves hypothesis 1. Only the mechanism proposed in hypothesis 3 with two binding modes and concerted decarboxylation with inversion of the stereocenter agrees with our experimental results. For stereoelectronic reasons a 180° arrangement of the incoming C74-SH proton and the leaving carboxyl group across the stereogenic carbon (Figure ) appears necessary to ensure optimal overlap between the participating orbitals of the S-H and C-C bonds. This consideration prompted us to use QM/MM well-tempered metadynamics for the simulation of potential alternative binding modes of 2a in AMDase ICPLLG that place the leaving carboxylate in a straight line with the catalytic C74 and the Ca.