Role of Tryptophan 38 in Loading Substrate Chain into the Active-site Tunnel of Cellobiohydrolase Ⅰ from Trichoderma reesei
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- Nakamura Akihiko
- Department of Applied Life Sciences, Faculty of Agriculture, Shizuoka University
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- Kanazawa Takashi
- School of Life Science and Technology, Tokyo Institute of Technology
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- Furuta Tadaomi
- School of Life Science and Technology, Tokyo Institute of Technology
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- Sakurai Minoru
- School of Life Science and Technology, Tokyo Institute of Technology
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- Saloheimo Markku
- VTT Technical Research Centre of Finland Ltd.
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- Samejima Masahiro
- Faculty of Engineering, Shinshu University Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo
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- Koivula Anu
- VTT Technical Research Centre of Finland Ltd.
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- Igarashi Kiyohiko
- VTT Technical Research Centre of Finland Ltd. Department of Biomaterials Sciences, Graduate School of Agricultural and Life Sciences, University of Tokyo
書誌事項
- タイトル別名
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- Role of Tryptophan 38 in Loading Substrate Chain into the Active-site Tunnel of Cellobiohydrolase I from <i>Trichoderma reesei</i>
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抄録
<p>Cellobiohydrolase I from Trichoderma reesei (TrCel7A) is one of the best-studied cellulases, exhibiting high activity towards crystalline cellulose. Tryptophan residues at subsites -7 and -4 (Trp40 and Trp38 respectively) are located at the entrance and middle of the tunnel-like active site of TrCel7A, and are conserved among the GH family 7 cellobiohydrolases. Trp40 of TrCel7A is important for the recruitment of cellulose chain ends on the substrate surface, but the role of Trp38 is less clear. Comparison of the effects of W38A and W40A mutations on the binding energies of sugar units at the two subsites indicated that the contribution of Trp38 to the binding was greater than that of Trp40. In addition, the smooth gradient of binding energy was broken in W38A mutant. To clarify the importance of Trp38, the activities of TrCel7A WT and W38A towards crystalline cellulose and amorphous cellulose were compared. W38A was more active than WT towards amorphous cellulose, whereas its activity towards crystalline cellulose was only one-tenth of that of WT. To quantify the effect of mutation at subsite -4, we measured kinetic parameters of TrCel7A WT, W40A and W38A towards cello-oligosaccharides. All combinations of enzymes and substrates showed substrate inhibition, and comparison of the inhibition constants showed that the Trp38 residue increases the velocity of substrate intake (kon for forming productive complex) from the minus side of the subsites. These results indicate a key role of Trp38 residue in processively loading the reducing-end of cellulose chain into the catalytic tunnel.</p>
収録刊行物
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- Ｊｏｕｒｎａｌ ｏｆ Ａｐｐｌｉｅｄ Ｇｌｙｃｏｓｃｉｅｎｃｅ
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Ｊｏｕｒｎａｌ ｏｆ Ａｐｐｌｉｅｄ Ｇｌｙｃｏｓｃｉｅｎｃｅ 68 (1), 19-29, 2021-03-11
日本応用糖質科学会