Finally, and interestingly, as shown in Figure , similar to the HG3 series where evolution eliminates a non-productive substrate binding conformation in the transition from the initial design to HG3.17, 32 also here, our optimization trajectory from GNCA4-WT through to V4-4 (+ polypeptide segment) significantly reduces sampling of the "OUT" substrate conformation observed in GNCA4-WT. That is, while this conformation is dominant in the GNCA4-WT (although nearly 69/31 split), the proportion of the conformations equilibrates in GNCA4-12 (44/56). The situation is completely reverted for V4 and V4-4, for which we observe the "OUT" conformation in only 34% and 20%, respectively of our simulation time (vs. 70% of simulation time in GNCA4-WT). Again, the reduction of the IN/OUT proportion observed from GNCA4-12 to V4 corroborates the enhancement of the catalytic activity when the polypeptide is added to the C-terminus of the enzyme. As shown in Figure , the polypeptide segment in V4-4 closes over the active site and is held in place by a hydrogen bonding interaction between D229 and the R266 from the polypeptide segment. On the one hand, this interaction, which is maintained for 44% of simulation time (at an average distance of 2.92 ± 0.24 Å taking snapshots every 10 ps of 5 x 1 μs trajectories) is catalytically detrimental as this salt bridge would be expected to stabilize the D229 side chain and thus decrease its pKa. However, this detrimental effect appears to be offset by the fact that this interaction makes the active site more compact, reducing its volume to 443.21.1 ± 165.7 Å 3 in V4-4 compared to 700.9 ± 143.2 Å 3 (for which the D229-R266 interaction is just kept for 2% of the simulation time), 651.2.9 ± 115.4 Å 3 in GNCA4-12 and 1433.0.0 ± 132.8 Å 3 in GNCA4-WT (as measured using Pocket Volume Measurer (POVME) 3.0, see the Experimental Section).