Molecular Dynamics (MD) simulations were carried out using the GROMACS simulation package v. 2022.05, together with the ff14SB 29 force field and TIP3P water model for the parameters of protein atoms and water molecules, respectively. We used two initial crystal structures (PDB ID: 4HJP Parameters for the phosphoenzyme intermediate were obtained from prior work, see the Supporting Information of ref. . Point mutations were introduced into the wild-type structures using PyMol. Residues 315 to 317 in PDB ID: 4HJP were missing from the crystal structure, and were manually introduced by overlay with PDB ID: 4GRZ. Missing residues at the N-and C-termini were omitted from our simulations (Gly241, Ser242, Val525 and Gln526 in both structures, and Gln527 and Ser528 in PDB ID: 4GRZ 31 ). PROPKA 3.0 was used to check for anomalous pKa values for ionizable residues, and, based on this evaluation all systems were kept in their standard protonation states at physiological pH. The protein was then placed inside a truncated octahedral water box of TIP3P water molecules, extending 10Å from the solute in all directions, and the system was neutralized with Na + counterions.