Why Do PETases Struggle with Crystalline PET? Catalytic Ensemble Sampling Reveals Molecular Bottlenecks

Polyethylene terephthalate (PET) is a widely used thermoplastic whose high crystallinity poses a major barrier to upscaling enzymatic recycling. Although PETases with high activity and stability have been reported, no enzyme has been shown to directly depolymerize crystalline PET (cPET), and the molecular determinants limiting their efficacy remain challenging to characterize. Here, we integrate experimental conformational ratios of crystalline and amorphous PET chains with enhanced-sampling molecular dynamics simulations to map the free-energy landscape of a prototypical PETase bound to PET oligomers, revealing how structural equilibria translate to catalytic function. We find that productive enzyme-substrate catalytic configurations can be reached for both crystalline and amorphous PET chains. However, the formation of catalytic ensembles on cPET is strongly hindered by the enzyme shape and dynamics, with additional energetic costs required to separate crystalline chains to fit the active site, consistent with experimental data. The model highlights limitations of the active-site architectures typically found in α/β-hydrolase scaffolds used for PET depolymerization and indicates directions for their redesign to enable cPET degradation. Our approach showcases a general strategy to leverage the quantitative characterization of catalytic ensembles for interrogating substrate-enzyme interactions and informing molecular design.