Interfacial molecular interactions of cellobiohydrolase Cel7A and its variants on cellulose

Background Molecular-scale mechanisms of the enzymatic breakdown of cellulosic biomass into fermentable sugars are still poorly understood, with a need for independent measurements of enzyme kinetic parameters. We measured binding times of cellobiohydrolase Trichoderma reesei Cel7A (Cel7A) on celluloses using wild-type Cel7A (WTintact), the catalytically deficient mutant Cel7A E212Q (E212Q(intact)) and their proteolytically isolated catalytic domains (CD) (WTcore and E212Q(core), respectively). The binding time distributions were obtained from time-resolved, super-resolution images of fluorescently labeled enzymes on cellulose obtained with total internal reflection fluorescence microscopy. Results Binding of WTintact and E212Q(intact) on the recalcitrant algal cellulose (AC) showed two bound populations: similar to 85% bound with shorter residence times of < 15 s while similar to 15% were effectively immobilized. The similarity between binding times of the WT and E212Q suggests that the single point mutation in the enzyme active site does not affect the thermodynamics of binding of this enzyme. The isolated catalytic domains, WTcore and E212Q(core), exhibited three binding populations on AC: similar to 75% bound with short residence times of similar to 15 s (similar to the intact enzymes), similar to 20% bound for < 100 s and similar to 5% that were effectively immobilized. Conclusions Cel7A binding to cellulose is driven by the interactions between the catalytic domain and cellulose. The cellulose-binding module (CBM) and linker increase the affinity of Cel7A to cellulose likely by facilitating recognition and complexation at the substrate interface. The increased affinity of Cel7A to cellulose by the CBM and linker comes at the cost of increasing the population of immobilized enzyme on cellulose. The residence time (or inversely the dissociation rates) of Cel7A on cellulose is not catalysis limited.