The Action of Plastic Degrading Enzyme is Accelerated Mainly Due to an Increase in Thermal Stability Rather Than by Inherent Catalytic Effect

Polyethylene terephthalate (PET) hydrolases offer a promising enzymatic route to plastic waste degradation under mild conditions. Among these, the engineered FAST-PETase variant exhibits superior catalytic efficiency and thermostability compared to the wild-type IsPETase, yet the molecular origins of these enhancements remain debated. In this work, we employ empirical valence bond (EVB) simulations in conjunction with semi-macroscopic PDLD/S-LRA calculations to investigate the rate-determining acylation step in PET dimer hydrolysis catalyzed by both wild-type and FAST-PETase. Our results successfully reproduce the experimentally observed trend in catalytic rate enhancement between the two systems. While prior interpretations attributing the improved activity to a strengthened hydrogen-bond network involving Asp106 and His237, we demonstrate that the distal N233K mutation in FAST-PETase induces long-range electrostatic changes that enhance catalytic efficiency by modulating the active site dipolar environment. More importantly, we show that the elevated performance of FAST-PETase at higher temperatures is not due to reduced flexibility in the mutant region but arises from enhanced thermal stability, which allows the enzyme to operate effectively at elevated temperatures and thus accelerate reaction rates. These findings underscore the central role of electrostatics and stability in enzyme engineering and suggest that data-driven methods, such as maximum entropy models, may enable the rational identification of further stability-enhancing mutations for improved PET depolymerization.