A simple yet cheap and efficient method to overcome this problem, consists in applying free energy perturbation (FEP) in combination with metadynamics simulations. This approach, known as MetaFEP, achieves the accuracy of the target level of theory requiring only a relatively small number of single-point calculations on the expensive high-level potential energy surface, reducing the computational cost but ensuring the sampling of the configurational space. This allows validation of the mechanistic hypothesis provided by the low-level method used for sampling, DFTB3 in this work, by comparing the FE profile with those obtained using MetaFEP with higher-level methods, DFT or MP2, and checking the consistency of the hypothesis. Finally, an atomistic explanation of the different reactivity observed for the two substrates from an energetic point of view is provided by the reactivity descriptors defined in the framework of the conceptual DFT . By providing the first molecular-level comparison of the reaction mechanism of PET and PEF hydrolysis, this study offers new insights to expand the applicability of this enzyme to a wider range of semi-aromatic plastic materials of industrial interest. Scheme 1. Representation of the PET hydrolysis mechanism by PETase. The scheme is represented using the PET4 model as a substrate. In each panel, the amino acids in the active site are shown: the catalytic triad (S160, H237, D206) is indicated in red, whereas W185, Y87, M161 and I208 are in purple. (A) The reaction starts with the deprotonation of S160 by H237 and the nucleophilic attack of the PET4 ester group. (B) A negatively charged tetrahedral transition state is formed, stabilized by the oxyanion hole formed by Y87 and M161 backbones. (C) The TS decomposes and forms the acyl-enzyme intermediate (S160-PET2''), completing the acylation stage. The first product of the hydrolysis leaves the catalytic domain (PET2'), followed by the entry of a water molecule within the binding pocket, which triggers the deacylation. (D) The reaction proceeds with the formation of a second negatively charged TS, stabilized by the oxyanion hole. Then, the second product of depolymerization is formed (PET2''), the enzyme is restored to its initial state and ready to restart the cycle with the binding of a new chain of PET.