As described before, HLDA requires as an input the equilibrium distributions of the selected features of each stable endpoint of the transition. These are collected from short, unbiased QM/MM MD simulations of reactant and products of both acylation and deacylation steps. For the acylation, we considered as reactant the system including enzyme and the substrate (PET4 or PEF4) and as a product the acyl-enzyme intermediate and the first leaving product (PET2' or PEF2'). For the deacylation, the acyl-enzyme intermediate was the reactant and the product was the enzyme with the hydrolyzed substrate (PET2' + PET2'' or PEF2' + PEF2''). The QM region was composed of the S160, H237, D206 sidechains and the substrate (PET4 or PEF4), counting a total of 115 and 100 atoms for the PETase-PET4 and PETase-PEF4 complexes, respectively, and a net charge of -1. Moreover, one water molecule was included for the simulation of the endpoint states of the deacylation step to react with the acyl-enzyme intermediate in the active site. The particle mesh Ewald (PME) method was employed to treat long-range electrostatic interactions, with a cutoff of 9 Å and a B-spline interpolation order 4. The electrostatic embedding scheme was used for the QM region adopting a cutoff of 12 Å. The covalent bonds truncated at the interface between the QM and MM regions were replaced with hydrogen link atoms (3 in total). Each system was relaxed using 500 steps of steepest descent followed by 500 steps of conjugate gradient method with the protein backbone restrained. Afterwards, 50 ps of equilibration were carried out in the canonical (NVT) ensemble heating the system from 0 K to 303.15 K with a heating rate of ~ 0.26 K • fs -1 . We then performed 100 ps of production run in the NPT ensemble at the pressure and temperatures of 1 bar and 303.15 K, realized with the Berendsen barostat 55 and the Langevin thermostat. The collision frequency and time constant were set to 2 ps -1 and 1 ps, respectively, for the Langevin dynamics, while a pressure relaxation time of 1 ps was used for the pressure regulation.