QM/MM-MD Studies on the Degradation Mechanism and Size Effect of PET by PETase

Polyethylene terephthalate (PET) has been widely used in our daily life, resulting in substantial accumulation of PET waste in the natural environment. PETase, in collaboration with MHETase, can effectively hydrolyze PET back into its constituent monomers, offering a promising solution for PET biorecycling. In this work, to address the controversial issues regarding the mechanism, the decomposition of PET oligomers by PETase was studied on the atomic level using M06-2X/MM-MD simulations. The reaction comprises two main stages: acylation and deacylation, each proceeding stepwise via a metastable intermediate. Deacylation is the rate-limiting step. The role of the third catalytic residue Asp177 was reinvestigated, which was found to take a combined charge-relay and low-energy hydrogen barrier mechanism to stabilize the tetrahedral transition states and intermediates. In addition, the influences of PET size on depolymerization activity were clarified, which enabled us to establish a relationship between structural features and the activation energy barrier. Ultimately, we have identified specific residues whose mutation could potentially enhance the enzyme’s activity based on the electrostatic interaction. This work not only provides valuable insights into the PETase catalytic mechanism but also lays a foundation for rational enzyme engineering strategies of PETase.