Single-molecule Imaging Analysis of Elementary Reaction Steps of Trichoderma reesei Cellobiohydrolase I ( Cel7A) Hydrolyzing Crystalline Cellulose I α and III I

Background: The catalytic mechanism of Trichoderma reesei cellobiohydrolase I (TrCel7A) is still unclear. Results:TrCel7A exhibited similar reaction kinetics during crystalline cellulose I and IIII hydrolysis. Conclusion: Not differences in kinetic parameters but surface properties of the crystalline cellulose influence the susceptibilities of cellulose I and IIII to hydrolysis by TrCel7A. Significance: Single-molecule measurements further our understanding of TrCel7A mechanism. Trichoderma reesei cellobiohydrolase I (TrCel7A) is a molecular motor that directly hydrolyzes crystalline celluloses into water-soluble cellobioses. It has recently drawn attention as a tool that could be used to convert cellulosic materials into biofuel. However, detailed mechanisms of action, including elementary reaction steps such as binding, processive hydrolysis, and dissociation, have not been thoroughly explored because of the inherent challenges associated with monitoring reactions occurring at the solid/liquid interface. The crystalline cellulose I and IIII were previously reported as substrates with different crystalline forms and different susceptibilities to hydrolysis by TrCel7A. In this study, we observed that different susceptibilities of cellulose I and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose I and IIII. Using single-molecule fluorescence microscopy and high speed atomic force microscopy, we also determined kinetic constants of the elementary reaction steps for TrCel7A against cellulose I and IIII. These measurements were performed at picomolar enzyme concentration in which density of TrCel7A on crystalline cellulose was very low. Under this condition, TrCel7A displayed similar binding and dissociation rate constants for cellulose I and IIII and similar fractions of productive binding on cellulose I and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose I and IIII with similar translational rates. With structural models of cellulose I and IIII, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lanes.