Mutation of the Y536 also has a severe impact on enzyme activity (0.6% of the WT). In our modelling, G540 took over the role of Y536 in the stabilization of substrate binding, forming an H-bond with a 3-keto group (d(C3=O---HN-G540 is 2.1 Å, Figure ). The position of the substrate was very similar to the WT and the proton abstraction turned out to be very easy (TS1 of 9.85 kcal/mol, Figure ). However, upon deprotonation, the conformation of the ring A in the substrate changed, shifting the C3=O group away from G540 and breaking the H-bond. As a result, only two tyrosine residues are involved in the interaction with enolate resulting in a small stabilization of the intermediate (by 0.35 kcal/mol) and difficult hydride transfer (TS2 35 kcal/mol).