The FE curves obtained for the acylation of PET4 (Figure ) and PEF4 (Figure ) indicate substantial differences in the underlying mechanisms. The acylation of the PETase-PET4 system occurs through a single, concerted step, in which a FE barrier peaking at ≈ 21 kcal/mol is found at a CV value of ≈ -1.7 (Figure ). In the reactant state (R), localized at a CV value of ≈ -0.5, the catalytic S160 and the substrate PET4 do not form covalent bonds (Table , d1 = 2.80 ± 0.30 Å). When the process starts, the O𝛾 atom of S160 progressively approaches the ester C5 atom of PET4 (d1 = 1.53 ± 0.07 Å). Concurrently, the O𝛾-H𝛾 bond of S160 elongates (d3), and the proton is progressively abstracted by the H237 side chain (d4). The O5'' atom of PET4 points towards the M161 and Y87 backbones, at distances (Table , d12, d13) and angles with the amide N-H groups (Table , a1, a2) compatible with the formation of stabilizing hydrogen bonds (HBs), although O5'' remains farther from M161 (d12) compared to Y87 (d13). At the peak of the FE barrier, a tetrahedral transition state (TSr1) structure is formed. S160 is fully deprotonated, with the H𝛾 atom being transiently captured by H237. At this stage, the carbonyl O5'' atom of PET4 has moved closer to the M161 and Y87 backbones, which form the oxyanion hole stabilizing the negatively charged TS. Moving along negative values of the reaction coordinate, the TS evolves towards the product, that is the acylenzyme intermediate (INTac-enz). In this state, the covalent bond between the O𝛾 atom of S160 and the C5 atom of PET4 is formed (d1 = 1.38 ± 0.04 Å), whereas the ether C5-O5' bond of PET4 is broken (d2 = 3.50 ± 0.15 Å). At the same time, the H𝛾 of S160 has been transferred to the O5' atom of PET4 (d6 = 0.97 ± 0.03 Å), yielding the first product of the catalytic cycle, composed of two PET monomers (PET2'), accompanied by a reaction energy of ≈ 4.2 kcal/mol. The FE profile obtained for the acylation of the PETase-PEF4 system (Figure ) shows substantial differences from that of PET4. In this case, the process is characterized by two energy barriers of ≈ 10 kcal/mol and ≈ 2 kcal/mol at values of ≈ -0.7 and ≈ -2.2, respectively, along the reaction coordinate, in the midst of which a metastable intermediate is observed. Similarly to PET4 acylation, the reaction starts with the progressive approach of the O𝛾 atom of S160 to the C5 atom of PEF4 and the stretching of the O-H bond: d1 decreases and d3 increases. At variance with the PET4 acylation, when the first TS (TSr1) is formed, the serine residue is not fully deprotonated, but the H𝛾 atom is shared between the O𝛾 atom of S160 and the N𝜀 atom of H237. Beyond the first FE barrier, at CV values of ≈ -1.7, the system reaches a metastable configuration that correlates with a tetrahedral configuration (INTtetr) in which S160 has transferred the H𝛾 atom to H237. H237 remains stably protonated (d4 = 1.06 ± 0.03 Å), producing a zwitterionic system with a neat charge separation: a positively charged H237 and a negatively charged tetrahedral intermediate. In this state, the O5'' atom forms tighter HBs with the oxyanion hole (Table ).