title: Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII.
abstract: 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 Iα 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 Iα and IIII are highly dependent on enzyme concentration, and at nanomolar enzyme concentration, TrCel7A shows similar rates of hydrolysis against cellulose Iα 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 Iα 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 Iα and IIII and similar fractions of productive binding on cellulose Iα and IIII. Furthermore, once productively bound, TrCel7A processively hydrolyzes and moves along cellulose Iα and IIII with similar translational rates. With structural models of cellulose Iα 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.
authors.affiliation: From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Shibafuji
authors.affiliation: the Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Nakamura
authors.affiliation: the Department of Physics, College of Science and Engineering, Kanazawa University, Kanazawa 920-1192, Japan, the Bio-AFM Frontier Research Center, College of Science and Engineering, Kanazawa University, Kanazawa 920-1192, Japan, and.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Uchihashi
authors.affiliation: the Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
authors.forename: Naohisa
authors.initials: N
authors.lastname: Sugimoto
authors.affiliation: the Department of Physics, College of Science and Engineering, Kanazawa University, Kanazawa 920-1192, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Fukuda
authors.affiliation: the Department of Physics, College of Science and Engineering, Kanazawa University, Kanazawa 920-1192, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Watanabe
authors.affiliation: the Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Samejima
authors.affiliation: the Department of Physics, College of Science and Engineering, Kanazawa University, Kanazawa 920-1192, Japan, the Bio-AFM Frontier Research Center, College of Science and Engineering, Kanazawa University, Kanazawa 920-1192, Japan, and.
authors.forename: Toshio
authors.initials: T
authors.lastname: Ando
authors.affiliation: From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Noji
authors.affiliation: the VTT Technical Research Centre of Finland, Espoo, FI-02044 VTT, Finland.
authors.forename: Anu
authors.initials: A
authors.lastname: Koivula
authors.affiliation: the Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan.
authors.forename: Kiyohiko
authors.initials: K
authors.lastname: Igarashi
authors.affiliation: From the Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan, iino@appchem.t.u-tokyo.ac.jp.
authors.forename: Ryota
authors.initials: R
authors.lastname: Iino
mesh_terms: D002482:Cellulose / Q000737:chemistry* / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000378:metabolism*; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D018625:Microscopy, Atomic Force; D008856:Microscopy, Fluorescence; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: Biofuel; Cellobiohydrolase; Cellulase; Cellulose; Enzyme Kinetics; High Speed Atomic Force Microscopy; Molecular Motors; Single-molecule Biophysics; Single-molecule Fluorescence Imaging