The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175

Anu Koivula, Laura Ruohonen, G. Wohlfahrt, Tapani Reinikainen, Tuula Teeri, K. Piens, M. Claeyssens, M. Weber, A. Vasella, D. Becker, M.L. Sinnott, J.Y. Zou, G.J. Kleywegt, M. Szardenings, J. Ståhlberg, T.A. Jones

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other.
Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base.
The value of D2O(V) of 1.16 ± 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.
Original languageEnglish
Pages (from-to)10015-10024
JournalJournal of the American Chemical Society
Volume124
Issue number34
DOIs
Publication statusPublished - 2002
MoE publication typeA1 Journal article-refereed

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Cellulose 1,4-beta-Cellobiosidase
Trichoderma
Aspartic Acid
Catalytic Domain
Hydrogen bonds
Enzyme kinetics
Mutagenesis
Proton transfer
Acids
Protonation
X ray crystallography
Catalysis
Water
Electrostatics
Hydrolysis
Tunnels
Stabilization
Glycoside Hydrolases
X Ray Crystallography
Hydrogen Bonding

Keywords

  • crystalline cellulose degradation
  • beta-cellobiosyl fluorides
  • fusca endocellulase E2
  • humicola-insolens
  • angstrom resolution
  • alpha-cellobiosyl
  • endoglucanase-I
  • catalytic core
  • hydrolysis
  • family-6

Cite this

Koivula, Anu ; Ruohonen, Laura ; Wohlfahrt, G. ; Reinikainen, Tapani ; Teeri, Tuula ; Piens, K. ; Claeyssens, M. ; Weber, M. ; Vasella, A. ; Becker, D. ; Sinnott, M.L. ; Zou, J.Y. ; Kleywegt, G.J. ; Szardenings, M. ; Ståhlberg, J. ; Jones, T.A. / The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175. In: Journal of the American Chemical Society. 2002 ; Vol. 124, No. 34. pp. 10015-10024.
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title = "The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175",
abstract = "Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of D2O(V) of 1.16 ± 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.",
keywords = "crystalline cellulose degradation, beta-cellobiosyl fluorides, fusca endocellulase E2, humicola-insolens, angstrom resolution, alpha-cellobiosyl, endoglucanase-I, catalytic core, hydrolysis, family-6",
author = "Anu Koivula and Laura Ruohonen and G. Wohlfahrt and Tapani Reinikainen and Tuula Teeri and K. Piens and M. Claeyssens and M. Weber and A. Vasella and D. Becker and M.L. Sinnott and J.Y. Zou and G.J. Kleywegt and M. Szardenings and J. St{\aa}hlberg and T.A. Jones",
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doi = "10.1021/ja012659q",
language = "English",
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Koivula, A, Ruohonen, L, Wohlfahrt, G, Reinikainen, T, Teeri, T, Piens, K, Claeyssens, M, Weber, M, Vasella, A, Becker, D, Sinnott, ML, Zou, JY, Kleywegt, GJ, Szardenings, M, Ståhlberg, J & Jones, TA 2002, 'The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175', Journal of the American Chemical Society, vol. 124, no. 34, pp. 10015-10024. https://doi.org/10.1021/ja012659q

The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175. / Koivula, Anu; Ruohonen, Laura; Wohlfahrt, G.; Reinikainen, Tapani; Teeri, Tuula; Piens, K.; Claeyssens, M.; Weber, M.; Vasella, A.; Becker, D.; Sinnott, M.L.; Zou, J.Y.; Kleywegt, G.J.; Szardenings, M.; Ståhlberg, J.; Jones, T.A.

In: Journal of the American Chemical Society, Vol. 124, No. 34, 2002, p. 10015-10024.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - The active site of cellobiohydrolase Cel6A from Trichoderma reesei: the roles of aspartic acids D221 and D175

AU - Koivula, Anu

AU - Ruohonen, Laura

AU - Wohlfahrt, G.

AU - Reinikainen, Tapani

AU - Teeri, Tuula

AU - Piens, K.

AU - Claeyssens, M.

AU - Weber, M.

AU - Vasella, A.

AU - Becker, D.

AU - Sinnott, M.L.

AU - Zou, J.Y.

AU - Kleywegt, G.J.

AU - Szardenings, M.

AU - Ståhlberg, J.

AU - Jones, T.A.

PY - 2002

Y1 - 2002

N2 - Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of D2O(V) of 1.16 ± 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.

AB - Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of D2O(V) of 1.16 ± 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.

KW - crystalline cellulose degradation

KW - beta-cellobiosyl fluorides

KW - fusca endocellulase E2

KW - humicola-insolens

KW - angstrom resolution

KW - alpha-cellobiosyl

KW - endoglucanase-I

KW - catalytic core

KW - hydrolysis

KW - family-6

U2 - 10.1021/ja012659q

DO - 10.1021/ja012659q

M3 - Article

VL - 124

SP - 10015

EP - 10024

JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

SN - 0002-7863

IS - 34

ER -