Direct measurement of PET hydrolase interfacial kinetics reveals catalysis-independent surface remodelling

Abstract

How PET hydrolases engage solid plastic has been inferred almost entirely from soluble analogs and surfactant-stabilised nanoparticle suspensions, and reported affinities span orders of magnitude. Here we measure it directly, depositing thin amorphous PET films onto gold surface plasmon resonance chips and following enzyme binding to authentic polymer in real time. Four PET hydrolases, LCC, LCC-ICCG, IsPETase-EHA and TfCut2, all bind with nanomolar apparent affinity and surface residence half-lives of tens of minutes. LCC-ICCG dissociates at 4.0 x 10^-1, within twofold of its kcat measured on authentic PET, indicating that turnover is limited by disengagement rather than by ester hydrolysis. During association, we observe non-monophasic responses for these enzymes, including, for several, a signal fall while enzyme is still flowing over the surface. This decline persists in a catalytically inactivated variant, is absent in control proteins and a structurally homologous non-plastic degrading cutinase, and cannot arise from movement of the bound enzyme alone. The polymer surface must therefore be altered non-catalytically by PET hydrolase binding. The process runs at different rates for different enzymes on an identical film, is suppressed by dilute Triton X-100, and is modulated biphasically by PET degradation products. Altogether, we conclude that enzymatic PET depolymerisation involves a catalysis-independent chain-mobilisation step that soluble assays cannot report.