The Ideonella sakaiensis PETase enzyme catalyzes the depolymerization of polyethylene terephthalate (PET), a major plastic pollutant. The reaction mechanism involves a two-step serine hydrolase process mediated by a serine-histidine-aspartate catalytic triad. Using transition path sampling and likelihood maximization, researchers identified optimal reaction coordinates for the PETase enzyme. They found that deacylation is likely the rate-limiting step, with reaction coordinates involving nucleophilic attack, ester bond cleavage, and the "moving-histidine" mechanism. The flexibility of Trp185 promotes the reaction, explaining decreased activity in mutations that restrict its motion. The active site of PETase includes the catalytic triad (Ser160, His237, Asp206), with Trp185 interacting with the PET substrate via π-π interactions. The study used QM/MM simulations to elucidate the acylation and deacylation steps, revealing that the moving histidine mechanism is used for proton transfer, and that Asp206 supports His237 through hydrogen bonding. The oxyanion hole residues (Met161 and Tyr87) stabilize the PET carboxyl oxygen, and Trp185 and Tyr87 provide π-π stabilization to the PET substrate. The reorientation of Trp185 between reaction steps facilitates the reaction, with deacylation being the rate-limiting step compared to acylation. The study also investigated the conformational changes of Trp185, showing that it adopts different conformations during the reaction. The results indicate that the moving histidine mechanism is used for proton transfer, and that the reaction proceeds through a concerted mechanism without a metastable intermediate. The findings provide insights into the PETase reaction mechanism, which can be applied to the function of PET hydrolysis in other enzymes. The study highlights the importance of understanding the reaction mechanism for future enzyme engineering efforts in plastics bioconversion.The Ideonella sakaiensis PETase enzyme catalyzes the depolymerization of polyethylene terephthalate (PET), a major plastic pollutant. The reaction mechanism involves a two-step serine hydrolase process mediated by a serine-histidine-aspartate catalytic triad. Using transition path sampling and likelihood maximization, researchers identified optimal reaction coordinates for the PETase enzyme. They found that deacylation is likely the rate-limiting step, with reaction coordinates involving nucleophilic attack, ester bond cleavage, and the "moving-histidine" mechanism. The flexibility of Trp185 promotes the reaction, explaining decreased activity in mutations that restrict its motion. The active site of PETase includes the catalytic triad (Ser160, His237, Asp206), with Trp185 interacting with the PET substrate via π-π interactions. The study used QM/MM simulations to elucidate the acylation and deacylation steps, revealing that the moving histidine mechanism is used for proton transfer, and that Asp206 supports His237 through hydrogen bonding. The oxyanion hole residues (Met161 and Tyr87) stabilize the PET carboxyl oxygen, and Trp185 and Tyr87 provide π-π stabilization to the PET substrate. The reorientation of Trp185 between reaction steps facilitates the reaction, with deacylation being the rate-limiting step compared to acylation. The study also investigated the conformational changes of Trp185, showing that it adopts different conformations during the reaction. The results indicate that the moving histidine mechanism is used for proton transfer, and that the reaction proceeds through a concerted mechanism without a metastable intermediate. The findings provide insights into the PETase reaction mechanism, which can be applied to the function of PET hydrolysis in other enzymes. The study highlights the importance of understanding the reaction mechanism for future enzyme engineering efforts in plastics bioconversion.