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 Ia and IIII 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 IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Ia 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 Ia 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 Ia and IIII and similar fractions of productive binding on cellulose Ia and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Ia and IIII with similar translational rates. With structural models of cellulose Ia 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 lane.
Shibafuji, Y., Nakamura, A., Uchihashi, T., Sugimoto, N., Fukuda, S., Watanabe, H., Samejima, M., Ando, T., Noji, H., Koivula, A., Igarashi, K., & Iino, R. (2014). Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Ia and IIII. Journal of Biological Chemistry, 289(20), 14056-14065. https://doi.org/10.1074/jbc.M113.546085