The contributions of the catalytic domain and carbohydrate-binding module to the overall binding kinetics of Trichoderma reesei Cel7A on cellulose

Molecular scale mechanisms of the enzymatic break down of cellulosic biomass into simple fermentable sugars are still poorly understood.  Accurate estimates of the enzyme kinetic parameters will pave the way for commercial success of biofuels. Experiments to understand the contribution of the functional domains of the cellobiohydrolase Trichoderma reesei Cel7A towards binding to cellulose were conducted using the whole enzyme (TrCel7A) and the proteolytically-isolated catalytic domain (CD) (TrCel7ACD). The desorption rate constants were calculated from binding time distributions obtained from time-resolved, super-resolution images of individual fluorescently-labeled enzyme molecules on cellulose using total internal reflectance fluorescence microscopy. Binding of TrCel7A on two strikingly different cellulosic substrates, crystalline algal cellulose (AC) and amorphous Phosphoric Acid Swollen Cellulose (PASC), showed the presence of three types of binding – a large fraction having a very short residence time of 2-10 s, an intermediate fraction having the residence time 10-30 s and a small fraction having a residence time of >100 s. Experiments conducted with TrCel7ACD showed the presence of only two fractions (intermediate residence time and longer residence time) suggesting that these two fractions arise from active site binding by the CD. Further, the fraction of CD binding of TrCel7A on PASC was four times larger than that on AC. Binding of TrCel7A on AC in the presence of cellobiose, an inhibitor of cellobiohydrolase activity, showed a 3.5 fold increase in the fraction bound by the CD, however exhibited a slightly lower residence time (20-30%) compared to standard conditions.