Single-molecule Imaging Analysis of Binding, Processive Movement, and Dissociation of Cellobiohydrolase Trichoderma reesei Cel6A and Its Domains on Crystalline Cellulose.
Trichoderma reesei Cel6A (TrCel6A) is a cellobiohydrolase that hydrolyzes crystalline cellulose into cellobiose. Here we directly observed the reaction cycle (binding, surface movement, and dissociation) of single-molecule intact TrCel6A, isolated catalytic domain (CD), cellulose-binding module (CBM), and CBM and linker (CBM-linker) on crystalline cellulose IαThe CBM-linker showed a binding rate constant almost half that of intact TrCel6A, whereas those of the CD and CBM were only one-tenth of intact TrCel6A. These results indicate that the glycosylated linker region largely contributes to initial binding on crystalline cellulose. After binding, all samples showed slow and fast dissociations, likely caused by the two different bound states due to the heterogeneity of cellulose surface. The CBM showed much higher specificity to the high affinity site than to the low affinity site, whereas the CD did not, suggesting that the CBM leads the CD to the hydrophobic surface of crystalline cellulose. On the cellulose surface, intact molecules showed slow processive movements (8.8 ± 5.5 nm/s) and fast diffusional movements (30-40 nm/s), whereas the CBM-Linker, CD, and a catalytically inactive full-length mutant showed only fast diffusional movements. These results suggest that both direct binding and surface diffusion contribute to searching of the hydrolysable point of cellulose chains. The duration time constant for the processive movement was 7.7 s, and processivity was estimated as 68 ± 42. Our results reveal the role of each domain in the elementary steps of the reaction cycle and provide the first direct evidence of the processive movement of TrCel6A on crystalline cellulose.
From the Okazaki Institute for Integrative Bioscience and.|the Department of Functional Molecular Science, School of Physical Sciences, Graduate University for Advanced Studies (SOKENDAI), Kanagawa 240-0193, Japan.
Akihiko
A
Nakamura
the Department of Applied Chemistry, Graduate School of Engineering, University of Tokyo, Tokyo 113-8656, Japan.
Tomoyuki
T
Tasaki
From the Okazaki Institute for Integrative Bioscience and.
Daiki
D
Ishiwata
From the Okazaki Institute for Integrative Bioscience and.
Mayuko
M
Yamamoto
From the Okazaki Institute for Integrative Bioscience and.
Yasuko
Y
Okuni
the Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
Akasit
A
Visootsat
the National Chemical Engineering Institute in Paris, Paris 75005, France.
Morice
M
Maximilien
the Department of Applied Chemistry, Graduate School of Engineering, University of Tokyo, Tokyo 113-8656, Japan.
Hiroyuki
H
Noji
the Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan, and.
Taku
T
Uchiyama
the Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan, and.
Masahiro
M
Samejima
the Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo 113-8657, Japan, and.|the VTT Technical Research Centre of Finland, Espoo FI-02044 VTT, Finland.
Kiyohiko
K
Igarashi
From the Okazaki Institute for Integrative Bioscience and iino@ims.ac.jp.|the Department of Functional Molecular Science, School of Physical Sciences, Graduate University for Advanced Studies (SOKENDAI), Kanagawa 240-0193, Japan.|Institute for Molecular Science, National Institutes of Natural Sciences, Aichi 444-8787, Japan.
Ryota
R
Iino
D002482:Cellulose / Q000737:chemistry*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry* / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D000072417:Protein Domains; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D005656:Fungal Proteins; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase
cellulase; cellulose; enzyme kinetics; enzyme mechanism; microscopic imaging; molecular motor; processivity; protein domain; single-molecule biophysics