The starting configurations of the PETase-PET4 and PETase-PEF4 complexes were chosen from the equilibrated structures produced in our previous work. In that study, the initial structure of the enzyme was selected from the protein databank (PDB ID: 5XJH ), while the PET4 and PEF4 chains were generated using the CHARMM-GUI Polymer Builder tool. The protonation state of charged amino acids was predicted with PropKa3 46,47 for reference physiological pH = 7, consistently with previous works. As a first step, the coordinate files produced with the NAMD software were converted into AMBER-compatible files in order to perform the MD simulations with the sander engine. This software was used because of the native implementations of the DFTB3 level of theory as well as the straightforward interface with other programs, including ORCA, 50 enabling the application of different QM methods. Therefore, the ligands were parametrized (PET4 or PEF4) using the antechamber suite included in AmberTools. The AM1-BCC charges and the gaff force field were used, writing out the missing parameters with the parmchk package. Then, the solvent and seven neutralizing Cl -ions were added, and the topology file was built with tleap. The ff14SB parameters were adopted for the protein and the TIP3P model for water, with a buffer region of at least 14 Å from any atom of the protein or the substrate to the edges of the simulation box. During this step, the disulfide bonds between cysteines C203 and C239 and between C273 and C289 of PETase were preserved. The final systems were inscribed inside rectangular boxes with size (80 x 80 x 70) Å 3 , counting approximately 43,000 atoms each.