However, probably the most interesting of all the active site residues is Trp331.

The authors have scanned potential energy along a single predefined reaction coordinate, using a very modest quantum-chemical description of the active site , namely the Becke-Perdew pure density functional together with a small basis (SVP) and a small quantum region (80 atoms).

Visual inspection of the active site showed near perfect overlap of the UDP molecules and neighboring side chains, allowing the coordinates of GalNAc to be directly transferred from 2D7I into 2FFU.

Furthermore, water molecules present in the active site were manually rotated to create a network of hydrogen bonds where possible.

Three of these water molecules are located close to the metal ion with one of them directly serving as a ligand and the other two forming a hydrogen bond network between the first water molecule and neighboring active site residues.

The native enzyme structure contains a manganese ion in the active site , coordinating the diphosphate fragment of UDP.

[26] Because this fact would entail a spin-unrestrict-ed treatment of the active site , leading to an almost twofold increase in computational cost and possible convergence problems, we opted for replacing it with magnesium.

The most stabilizing point mutation was D27F, which is located in the active site of the protein, rendering it inactive.

However for the rest of mutations outside of the active site we observed a weak yet statistically significant positive correlation between thermal stability and catalytic activity indicating the lack of a stability-activity tradeoff for DHFR.

This conclusion was reached in [53,58], based on the exploration of stability effects of mutations in the active site of beta-lactamase [53] and rubisco [58].

Fersht and coauthors also found several stabilizing mutations in the active site of Barnase rendering the protein inactive [59].

9 shows that exploring only mutations in the active site provides a biased view on the tradeoff between activity and stability.

Our data, however, paints a different picture for DHFR—of a weak positive correlation between Tm and kcat or kcat/KM (r = 0.46, p = 0.02 and r = 0.41, p = 0.03 respectively) with one notable outlier D27F, where the stabilizing mutation is made right in the active site (Fig.