The active site of Trichoderma reesei cellobiohydrolase II: The role of tyrosine 169

Anu Koivula (Corresponding Author), Tapani Reinikainen, Laura Ruohonen, Anne Valkeajärvi, Marc Claeyssens, Olle Teleman, Gerard Kleywegt, Michael Szardenings, Juha Rouvinen, Alwyn Jones (Corresponding Author), Tuula Teeri

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Abstract

Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The β-l,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified experimentally. The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis. The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding behaviour of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 Å resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have asecond role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.
Original languageEnglish
Pages (from-to)691-699
Number of pages9
JournalProtein Engineering
Volume9
Issue number8
DOIs
Publication statusPublished - 1996
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Catalysis
Tyrosine
Catalytic Domain
Protonation
Cellobiose
Glucose
Enzymes
Crystal structure
Aspartic Acid
Reaction intermediates
Acids
Conformations
Cellulose
Tunnels
Protons
Association reactions
Kinetics
Mutation

Cite this

Koivula, A., Reinikainen, T., Ruohonen, L., Valkeajärvi, A., Claeyssens, M., Teleman, O., ... Teeri, T. (1996). The active site of Trichoderma reesei cellobiohydrolase II: The role of tyrosine 169. Protein Engineering, 9(8), 691-699. https://doi.org/10.1093/protein/9.8.691
Koivula, Anu ; Reinikainen, Tapani ; Ruohonen, Laura ; Valkeajärvi, Anne ; Claeyssens, Marc ; Teleman, Olle ; Kleywegt, Gerard ; Szardenings, Michael ; Rouvinen, Juha ; Jones, Alwyn ; Teeri, Tuula. / The active site of Trichoderma reesei cellobiohydrolase II : The role of tyrosine 169. In: Protein Engineering. 1996 ; Vol. 9, No. 8. pp. 691-699.
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title = "The active site of Trichoderma reesei cellobiohydrolase II: The role of tyrosine 169",
abstract = "Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The β-l,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified experimentally. The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis. The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding behaviour of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 {\AA} resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have asecond role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.",
author = "Anu Koivula and Tapani Reinikainen and Laura Ruohonen and Anne Valkeaj{\"a}rvi and Marc Claeyssens and Olle Teleman and Gerard Kleywegt and Michael Szardenings and Juha Rouvinen and Alwyn Jones and Tuula Teeri",
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Koivula, A, Reinikainen, T, Ruohonen, L, Valkeajärvi, A, Claeyssens, M, Teleman, O, Kleywegt, G, Szardenings, M, Rouvinen, J, Jones, A & Teeri, T 1996, 'The active site of Trichoderma reesei cellobiohydrolase II: The role of tyrosine 169', Protein Engineering, vol. 9, no. 8, pp. 691-699. https://doi.org/10.1093/protein/9.8.691

The active site of Trichoderma reesei cellobiohydrolase II : The role of tyrosine 169. / Koivula, Anu (Corresponding Author); Reinikainen, Tapani; Ruohonen, Laura; Valkeajärvi, Anne; Claeyssens, Marc; Teleman, Olle; Kleywegt, Gerard; Szardenings, Michael; Rouvinen, Juha; Jones, Alwyn (Corresponding Author); Teeri, Tuula.

In: Protein Engineering, Vol. 9, No. 8, 1996, p. 691-699.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - The active site of Trichoderma reesei cellobiohydrolase II

T2 - The role of tyrosine 169

AU - Koivula, Anu

AU - Reinikainen, Tapani

AU - Ruohonen, Laura

AU - Valkeajärvi, Anne

AU - Claeyssens, Marc

AU - Teleman, Olle

AU - Kleywegt, Gerard

AU - Szardenings, Michael

AU - Rouvinen, Juha

AU - Jones, Alwyn

AU - Teeri, Tuula

N1 - Project code: BEL4319

PY - 1996

Y1 - 1996

N2 - Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The β-l,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified experimentally. The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis. The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding behaviour of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 Å resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have asecond role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.

AB - Trichoderma reesei cellobiohydrolase II (CBHII) is an exoglucanase cleaving primarily cellobiose units from the non-reducing end of cellulose chains. The β-l,4 glycosidic bond is cleaved by acid catalysis with an aspartic acid, D221, as the likely proton donor, and another aspartate, D175, probably ensuring its protonation and stabilizing charged reaction intermediates. The catalytic base has not yet been identified experimentally. The refined crystal structure of CBHII also shows a tyrosine residue, Y169, located close enough to the scissile bond to be involved in catalysis. The role of this residue has been studied by introducing a mutation Y169F, and analysing the kinetic and binding behaviour of the mutated CBHII. The crystal structure of the mutated enzyme was determined to 2.0 Å resolution showing no changes when compared with the structure of native CBHII. However, the association constants of the mutant enzyme for cellobiose and cellotriose are increased threefold and for 4-methylumbelliferyl cellobioside over 50-fold. The catalytic constants towards cellotriose and cellotetraose are four times lower for the mutant. These data suggest that Y169, on interacting with a glucose ring entering the second subsite in a narrow tunnel, helps to distort the glucose ring into a more reactive conformation. In addition, a change in the pH activity profile was observed. This indicates that Y169 may have asecond role in the catalysis, namely to affect the protonation state of the active site carboxylates, D175 and D221.

U2 - 10.1093/protein/9.8.691

DO - 10.1093/protein/9.8.691

M3 - Article

VL - 9

SP - 691

EP - 699

JO - Protein Engineering, Design and Selection

JF - Protein Engineering, Design and Selection

SN - 1741-0126

IS - 8

ER -