Rate‐limiting step and substrate accessibility of cellobiohydrolase Cel6A from Trichoderma reesei

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

The cellobiohydrolase (CBH) Cel6A is an important component of enzyme cocktails for industrial degradation of lignocellulosic biomass. However, the kinetics of this enzyme acting on its natural, insoluble substrate remains sparsely investigated. Here, we studied Cel6A from Trichoderma reesei with respect to adsorption, processivity, and kinetics both in the steady-state and pre-steady-state regimes, on microcrystalline and amorphous cellulose. We found that slow dissociation (koff ) was limiting the overall reaction rate, and we suggest that this leads to an accumulation of catalytically inactive complexes in front of obstacles and irregularities on the cellulose surface. The processivity number of Cel6A was low on both investigated substrates (5-10), and this suggested a rugged surface with short obstacle-free path lengths. The turnover of the inner catalytic cycle (the reactions of catalysis in one processive step) was too fast to be fully resolved, but a minimum value of about 20 s-1 could be established. This is among the highest values reported hitherto for a cellulase, and it underscores the catalytic efficiency of Cel6A. Conversely, we found that Cel6A had a poor ability to recognize attack sites on the cellulose surface. On amorphous cellulose, for example, Cel6A was only able to initiate hydrolysis on about 4% of the sites to which it could adsorb. This probably reflects high requirements of Cel6A to the architecture of the site. We conclude that compared to the other CBH, Cel7A, secreted by T. reesei, Cel6A is catalytically more efficient but less capable of attacking a broad range of structurally distinct sites on the cellulose surface. ENZYMES: TrCel6A, nonreducing end-acting cellobiohydrolase (EC3.2.1.91) from Trichoderma reesei; TrCel7A, reducing end-acting cellobiohydrolase (EC3.2.1.176) from T. reesei.
OriginalsprogEngelsk
TidsskriftF E B S Journal
Vol/bind285
Udgave nummer23
Sider (fra-til)4482-4493
Antal sider12
ISSN1742-464X
DOI
StatusUdgivet - 17 okt. 2018

Bibliografisk note

"This is the peer reviewed version of the following article: Christensen, S. J., Kari, J. , Badino, S. F., Borch, K. and Westh, P. (2018), Rate‐limiting step and substrate accessibility of cellobiohydrolase Cel6A from Trichoderma reesei. FEBS J, 285: 4482-4493. doi:10.1111/febs.14668, which has been published in final form at https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.14668. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions."

Emneord

  • Cel6A
  • Cellobiohydrolases
  • Cellulose
  • Enzyme kinetics
  • Quenched-flow

Citer dette

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title = "Rate‐limiting step and substrate accessibility of cellobiohydrolase Cel6A from Trichoderma reesei",
abstract = "The cellobiohydrolase (CBH) Cel6A is an important component of enzyme cocktails for industrial degradation of lignocellulosic biomass. However, the kinetics of this enzyme acting on its natural, insoluble substrate remains sparsely investigated. Here, we studied Cel6A from Trichoderma reesei with respect to adsorption, processivity, and kinetics both in the steady-state and pre-steady-state regimes, on microcrystalline and amorphous cellulose. We found that slow dissociation (koff ) was limiting the overall reaction rate, and we suggest that this leads to an accumulation of catalytically inactive complexes in front of obstacles and irregularities on the cellulose surface. The processivity number of Cel6A was low on both investigated substrates (5-10), and this suggested a rugged surface with short obstacle-free path lengths. The turnover of the inner catalytic cycle (the reactions of catalysis in one processive step) was too fast to be fully resolved, but a minimum value of about 20 s-1 could be established. This is among the highest values reported hitherto for a cellulase, and it underscores the catalytic efficiency of Cel6A. Conversely, we found that Cel6A had a poor ability to recognize attack sites on the cellulose surface. On amorphous cellulose, for example, Cel6A was only able to initiate hydrolysis on about 4{\%} of the sites to which it could adsorb. This probably reflects high requirements of Cel6A to the architecture of the site. We conclude that compared to the other CBH, Cel7A, secreted by T. reesei, Cel6A is catalytically more efficient but less capable of attacking a broad range of structurally distinct sites on the cellulose surface. ENZYMES: TrCel6A, nonreducing end-acting cellobiohydrolase (EC3.2.1.91) from Trichoderma reesei; TrCel7A, reducing end-acting cellobiohydrolase (EC3.2.1.176) from T. reesei.",
keywords = "Cel6A, Cellobiohydrolases, Cellulose, Enzyme kinetics, Quenched-flow, Cel6A, cellobiohydrolases, cellulose, enzyme kinetics, quenched-flow",
author = "Christensen, {Stefan Jarl} and Jeppe Kari and Badino, {Silke Flindt} and Kim Borch and Peter Westh",
note = "{"}This is the peer reviewed version of the following article: Christensen, S. J., Kari, J. , Badino, S. F., Borch, K. and Westh, P. (2018), Rate‐limiting step and substrate accessibility of cellobiohydrolase Cel6A from Trichoderma reesei. FEBS J, 285: 4482-4493. doi:10.1111/febs.14668, which has been published in final form at https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.14668. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.{"}",
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Rate‐limiting step and substrate accessibility of cellobiohydrolase Cel6A from Trichoderma reesei. / Christensen, Stefan Jarl; Kari, Jeppe; Badino, Silke Flindt; Borch, Kim; Westh, Peter.

I: F E B S Journal, Bind 285, Nr. 23, 17.10.2018, s. 4482-4493.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Rate‐limiting step and substrate accessibility of cellobiohydrolase Cel6A from Trichoderma reesei

AU - Christensen, Stefan Jarl

AU - Kari, Jeppe

AU - Badino, Silke Flindt

AU - Borch, Kim

AU - Westh, Peter

N1 - "This is the peer reviewed version of the following article: Christensen, S. J., Kari, J. , Badino, S. F., Borch, K. and Westh, P. (2018), Rate‐limiting step and substrate accessibility of cellobiohydrolase Cel6A from Trichoderma reesei. FEBS J, 285: 4482-4493. doi:10.1111/febs.14668, which has been published in final form at https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.14668. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions."

PY - 2018/10/17

Y1 - 2018/10/17

N2 - The cellobiohydrolase (CBH) Cel6A is an important component of enzyme cocktails for industrial degradation of lignocellulosic biomass. However, the kinetics of this enzyme acting on its natural, insoluble substrate remains sparsely investigated. Here, we studied Cel6A from Trichoderma reesei with respect to adsorption, processivity, and kinetics both in the steady-state and pre-steady-state regimes, on microcrystalline and amorphous cellulose. We found that slow dissociation (koff ) was limiting the overall reaction rate, and we suggest that this leads to an accumulation of catalytically inactive complexes in front of obstacles and irregularities on the cellulose surface. The processivity number of Cel6A was low on both investigated substrates (5-10), and this suggested a rugged surface with short obstacle-free path lengths. The turnover of the inner catalytic cycle (the reactions of catalysis in one processive step) was too fast to be fully resolved, but a minimum value of about 20 s-1 could be established. This is among the highest values reported hitherto for a cellulase, and it underscores the catalytic efficiency of Cel6A. Conversely, we found that Cel6A had a poor ability to recognize attack sites on the cellulose surface. On amorphous cellulose, for example, Cel6A was only able to initiate hydrolysis on about 4% of the sites to which it could adsorb. This probably reflects high requirements of Cel6A to the architecture of the site. We conclude that compared to the other CBH, Cel7A, secreted by T. reesei, Cel6A is catalytically more efficient but less capable of attacking a broad range of structurally distinct sites on the cellulose surface. ENZYMES: TrCel6A, nonreducing end-acting cellobiohydrolase (EC3.2.1.91) from Trichoderma reesei; TrCel7A, reducing end-acting cellobiohydrolase (EC3.2.1.176) from T. reesei.

AB - The cellobiohydrolase (CBH) Cel6A is an important component of enzyme cocktails for industrial degradation of lignocellulosic biomass. However, the kinetics of this enzyme acting on its natural, insoluble substrate remains sparsely investigated. Here, we studied Cel6A from Trichoderma reesei with respect to adsorption, processivity, and kinetics both in the steady-state and pre-steady-state regimes, on microcrystalline and amorphous cellulose. We found that slow dissociation (koff ) was limiting the overall reaction rate, and we suggest that this leads to an accumulation of catalytically inactive complexes in front of obstacles and irregularities on the cellulose surface. The processivity number of Cel6A was low on both investigated substrates (5-10), and this suggested a rugged surface with short obstacle-free path lengths. The turnover of the inner catalytic cycle (the reactions of catalysis in one processive step) was too fast to be fully resolved, but a minimum value of about 20 s-1 could be established. This is among the highest values reported hitherto for a cellulase, and it underscores the catalytic efficiency of Cel6A. Conversely, we found that Cel6A had a poor ability to recognize attack sites on the cellulose surface. On amorphous cellulose, for example, Cel6A was only able to initiate hydrolysis on about 4% of the sites to which it could adsorb. This probably reflects high requirements of Cel6A to the architecture of the site. We conclude that compared to the other CBH, Cel7A, secreted by T. reesei, Cel6A is catalytically more efficient but less capable of attacking a broad range of structurally distinct sites on the cellulose surface. ENZYMES: TrCel6A, nonreducing end-acting cellobiohydrolase (EC3.2.1.91) from Trichoderma reesei; TrCel7A, reducing end-acting cellobiohydrolase (EC3.2.1.176) from T. reesei.

KW - Cel6A

KW - Cellobiohydrolases

KW - Cellulose

KW - Enzyme kinetics

KW - Quenched-flow

KW - Cel6A

KW - cellobiohydrolases

KW - cellulose

KW - enzyme kinetics

KW - quenched-flow

U2 - 10.1111/febs.14668

DO - 10.1111/febs.14668

M3 - Journal article

VL - 285

SP - 4482

EP - 4493

JO - F E B S Journal

JF - F E B S Journal

SN - 1742-464X

IS - 23

ER -