Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose I_a and III_I

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_a and III_I were previously reported as substrates with different crystalline formsanddifferent susceptibilities to hydrolysis byTrCel7A. In this study,weobserved that different susceptibilities of cellulose I_a and III_I are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose I_a and III_I. 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_a and III_I. 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_a and III_I and similar fractions of productive binding on cellulose Ia and III_I. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose I_a and III_I with similar translational rates. With structural models of cellulose I_a and III_I, we propose that different susceptibilities at high TrCel7A concentration arise from surface properties of substrate, including ratio of hydrophobic surface and number of available lane.