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Polyethylene terephthalate (PET), the most abundantly produced polyester plastic, can be depolymerized by the Ideonella sakaiensis PETase enzyme. Based on multiple PETase crystal structures, the reaction has been proposed to proceed via a two-step serine hydrolase mechanism mediated by a serine-histidine-aspartate catalytic triad. To elucidate the multi-step PETase catalytic mechanism, we use transition path sampling and likelihood maximization to identify optimal reaction coordinates for the PETase enzyme. We predict that deacylation is likely rate-limiting, and the reaction coordinates for both steps include elements describing nucleophilic attack, ester bond cleavage, and the "moving-histidine" mechanism. We find that the flexibility of Trp185 promotes the reaction, providing an explanation for decreased activity observed in mutations that restrict Trp185 motion. Overall, this study uses unbiased computational approaches to reveal the detailed reaction mechanism necessary for further engineering of an important class of enzymes for plastics bioconversion. Polyethylene terephthalate (PET) can be depolymerized by the Ideonella sakaiensis PETase enzyme, however, questions remain about the precise catalytic mechanism. Here, the authors use unbiased QM/MM MD simulations to determine optimal mechanistic descriptions of the acylation and deacylation reactions, revealing the rate-limiting step and key interactions within the catalytic triad and Trp185 conformation. [ABSTRACT FROM AUTHOR]