As expected, the Y363F mutation of AcmB that eliminates the tyrosyl residue from the active site renders the enzyme completely inactive (Tables and). The second most severe mutation is Y118F, which interrupts the H-bond with Y363. We were not able to determine the activity of the mutant enzyme in a spectrophotometric assay, but we detected the product after overnight incubation. The QM/MM modelling (Figure and S25) showed that the Y118F mutation facilitates proton removal from the substrate (TS1 lowered by 2.3 kcal/mol), but the enolate intermediate is less stable and the barrier of the hydride transfer is increased by 6.1 kcal/mol. This can be explained by an increased nucleophilic character of the Y363 ion which is not moderated by the H-bond with Y118, as depicted by a much lower charge on the Y363 oxygen atom in Y118F vs. WT enzyme (q APT -0.9 vs -0.46, respectively). However, the lack of an additional H-bond with Y118 and enolic intermediate's 3-keto group results in increased energy of E:I and TS2, due to worse enolate stabilization (i.e. higher charge of O atom bound to C3 and at C2 atom in Y118F than in the WT).