Enzyme FAST-PETase, recently obtained by machine learning approach, can depolymerize polyethylene terephthalate (PET), a synthetic resin employed in plastics and in clothing fibers. Therefore it represents a promising solution for the recycling of PET-based materials. In the present study, a model of PET was adopted to describe the substrate, and all-atoms classical molecular dynamics (MD) simulations on apo- and substrate bound- FAST-PETase were carried out at 30°C and 50°C, to provide atomistic details on the binding step of the catalytic cycle. Comparative analysis sheds light on the interactions occurring between the FAST-PETase and 4PET at 50 °C, the optimal working condition of the enzyme. Pre-organization of the enzyme active and binding sites has been highlighted while MD of FAST-PETase:4PET pointed out on the occurrence of solvent-inaccessible conformations of the substrate promoted by the enzyme. Indeed, neither of these conformations were observed during MD of substrate alone in solution performed at 30 °C, 50 °C and 150 °C. The analysis led us to propose that, at 50 °C, the FAST-PETase is pre-organized to bind the PET and that the interactions occurring in the binding site can promote more reactive conformation of PET substrate, thus enhancing the catalytic activity of the enzyme.