## 3 Results and Discussion
### IsPETase Activity on Terephthalate Aromatic Polyesters  
PET, PBT, PHT, and AkestraTM were used as substrates for IsPETase. In all cases, the main product was TPA (Figure 7), unlike previous reactions where MHET was the predominant product of PET depolymerization (Figure 3A). This could be due to the substantially higher amount of enzyme used for these substrates (0.01 mg IsPETase per mg of the substrate). The enzymatic products were quantified as TPAeq, and the percentage of depolymerization was calculated as described in Section 3.2. Thus, PET showed remarkable enzymatic depolymerization (39%) compared with the other polymers (<0.25%) (Table 3). Interestingly, although by far, the second-best substrate was PBT, followed by PHT and AkestraTM, suggesting a correlation of IsPETase activity with the length and size of the TPA linker group present on the polymer.  
The ligand binding modes were studied by coupling monomers BHET, BHBT, BHHT, and an AkestraTM fragment at subsite I of _Is_PETase (Figure 8). The complexes obtained were compared with the co-crystallized structure of the hydroxyethyl methyl terephthalate (HEMT)/_Is_PETase double mutant (PDB: 5 XH3). This mutant has glycine instead of arginine 132 and alanine instead of nucleophilic serine 160 (corresponding to R103G/S13A in 5 XH3) [23]. The coupled BHET, corresponding to a PET monomer, had the same pose as the co-crystallized HEMT analog (Figure 8A), indicating a reliable coupling prediction, and no further refinements were needed. Indeed, the aromatic and carbonyl groups of all the ligands overlapped very well with the corresponding groups of HEMT (Figure 8B-D). Therefore, the main interactions between the studied ligands and the subsite I were conserved. It was suggested that Trp 185 and Trp 159 form p-p interactions with the aromatic groups of the ligands at subsites I and II, respectively [17]. In the complexes obtained here, Trp 185 forms p-p interactions with all ligands. However, the length of the diol linkers in BHBT and BHHT seems unfavorable for an interaction of a second aromatic ring with Trp 159. Interestingly, one of the m-dioxane rings of the AkestraTM fragment formed a hydrophobic interaction with Trp 159 (Figure 8D), but the complexity of its polymeric form could significantly reduce the enzyme's access to ester linkages. Therefore, the low  
Figure 7: HPLC analysis of _Is_PETase depolymerization of terephthalate polyesters after 72 h: poly(ethylene) terephthalate (PET), poly(butylene) terephthalate (PBT), poly(hexamethylene) terephthalate (PHT), and AkestraTM.  
IsPETase activity in PBT, PHT, and AkestraTM could be attributed to limited access or unfavorable interactions at subsite II.  
Figure 8: Docking of different ligands (represented in green) into _Is_PETase (in cyan) subsite I. The whole active site is indicated with segmented lines. The hydrophobicity surfaces of the active sites are represented according to the Kyte-Doolittle scale, ranging from dodger blue for the most hydrophilic to white and to orange-red for the most hydrophobic [57]. Every obtained enzyme/ligand complex is overlapped with the co-crystallized structure HEMT/double mutant _Is_PETase (5 XH3), where the HEMT is represented in yellow and the mutant _Is_PETase is in light sea green. The catalytic amino acids are labeled in red. (**A**) BHET/_Is_PETase. (**B**) BHET/_Is_PETase. (**C**) BHFIT/_Is_PETase. (**D**) AkestraTM fragment/_Is_PETase.