Finally, the pKas of ionizable residues in all systems were predicted using PROPKA 3.0 to identify any amino acid side chains with potentially anomalous pKas. Based on this, in addition to the expected elevated pKa of the catalytic base D229, D246 and K234 were both predicted to have anomalous pKas (in the range of 8.7-11.0 for D246 and 6.3-6.5 for K234), which was confirmed to be reasonable by visual inspection of the local environment of these side chains, and thus these side chains were kept in their neutral states throughout our simulations. All other side chains were kept in their standard protonation states at physiological pH. All systems were solvated in a truncated octahedral water box of OPC water molecules, 73 extending 11.0Å from the protein in all directions, and all systems were neutralized using Na + counterions (8 for GNCA4-WT, 9 for GNCA4-12, and 10 for V4-4 with and without the polypeptide segment). Finally, all hydrogen atoms in the system were scaled using hydrogen mass repartitioning. Molecular Dynamics Simulations. All MD simulations in this work were performed using the CUDA-accelerated version of Amber22, 75 using the ff19SB force field, and the OPC water model. Partial charges for the substrate were calculated at the HF/6-31G(d) level of theory by restrained electrostatic potential (RESP) fitting using Antechamber, 78 based on gas-phase geometries optimized at the B3LYP/6-31G(d) level of theory, using Gaussian 16 Rev. B.01. All other force field parameters used to describe the substrate 5-nitrobenzisoxazole were obtained using the General AMBER Force Field (GAFF2), and all non-standard substrate parameters are provided for reference in Table , and in the Zenodo data package submitted at DOI:10.5281/zenodo.12666978.