Finally, we studied the mutation G540P, which aimed at removing the auxiliary H-bond between the main chain amino group and the 3-keto group of the substrate. However, structural interference introduced by the proline resulted in misfolding of the enzyme. Despite that, we were still able to detect trace activity after overnight incubation of the enzyme with the substrate. Unfortunately, we had to assume that the introduced mutation resulted in a severe change in the protein structure, which does not allow for the modelling of this effect nor gives any insight into the structure of the E:S complex.