Measuring productive and non-productive binding of cellobiohydrolase on cellulose by time-resolved, super-resolution single molecule imaging

Lignocellulosic biomass shows promise as a commercially viable feedstock for bioconversion to fuels and other bioproducts. At the heart of the process is the enzymatic saccharification of the highly recalcitrant cellulose with the goals of maximizing yield while minimizing reaction time. An understanding of cellulose hydrolysis mechanisms is critical to commercial success, but currently incomplete. Recent studies have shown that the cellobiohydrolase Trichoderma reesei Cel7A (TrCel7A) processively hydrolyze cellulose at an average speed of ~ 5 hydrolytic events per second for ~10 - 100 successive hydrolytic events when productively bound. TrCel7A bind to cellulose fibrils by two modes - the catalytic domain (CD) binds an isolated cellodextrin molecule in the active site by a combination of hydrophobic interactions and hydrogen bonding interactions while hydrophobic interactions mediate the association of the carbohydrate binding module (CBM) to the hydrophobic surfaces of microfibrils. The dissociation of TrCel7A bound via its CD has been postulated to limit the reaction, however literature estimates of the dissociation constant, k_off vary over several orders of magnitude. To gain further insight into the modes of binding of TrCel7A to cellulose, we have tracked the binding dynamics of individual TrCel7A molecules on the surface of cellulose fibrils by time-resolved, super-resolution single molecule imaging. We estimate enzyme dissociation rates attributed to CD and the CBM of TrCel7A by measuring single-molecule residency time distributions of TrCel7A and the catalytic domain of TrCel7A (TrCel7ACD) bound to cellulose to gain insight into the role of productive and non-productive binding.