As our starting point, we benchmarked our empirical valence bond (EVB) model by performing simulations of our baseline enzyme, GNCA4-WT, as well as six active site mutants: G62S, A146G, A173V, R256A, R256K, L265Q, described in the section Attempting to Increase De Novo Enzyme Activity Through Random Library Screening. As can be seen from Table , the effect of these mutations on the catalytic activity is minimal, with a mere 3.3-fold difference in kcat/KM (M -1 s -1 ) between the most and least active variants, an effect which is mainly caused by differences in KM. The kcat values are very similar, resulting in activation free energies that are within 0.3 kcal•mol -1 of each other across the series. Following from this, our EVB simulations were able to reproduce the experimental activation free energy for both the GNCA β-lactamase W290D-F290W to within 0.5 kcal•mol -1 (Figure and Table ). The correlation coefficient between the calculated and experimental activation free energies is -0.46, calculated using linear regression analysis (Figure ). This shows moderate correlation between the calculated and experimental values, taking into account that the differences in energies for each system are so small, and thus even very small thermodynamic differences can lead to weaker correlation with the experimental values.