Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

Nicolaj Cruys-Bagger, Jens Elmerdahl Olsen, Eigil Præstgaard, Hirosuke Tatsumi, Nikolaj Spodsberg, Kim Borch, Peter Westh

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10–25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ∼30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.
OriginalsprogEngelsk
TidsskriftJournal of Biological Chemistry
Vol/bind287
Udgave nummer22
Sider (fra-til)18451-18458
ISSN0021-9258
DOI
StatusUdgivet - 2012

Citer dette

Cruys-Bagger, N., Olsen, J. E., Præstgaard, E., Tatsumi, H., Spodsberg, N., Borch, K., & Westh, P. (2012). Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A. Journal of Biological Chemistry, 287(22), 18451-18458. https://doi.org/10.1074/jbc.M111.334946
Cruys-Bagger, Nicolaj ; Olsen, Jens Elmerdahl ; Præstgaard, Eigil ; Tatsumi, Hirosuke ; Spodsberg, Nikolaj ; Borch, Kim ; Westh, Peter. / Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A. I: Journal of Biological Chemistry. 2012 ; Bind 287, Nr. 22. s. 18451-18458.
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abstract = "The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10–25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ∼30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.",
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Cruys-Bagger, N, Olsen, JE, Præstgaard, E, Tatsumi, H, Spodsberg, N, Borch, K & Westh, P 2012, 'Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A', Journal of Biological Chemistry, bind 287, nr. 22, s. 18451-18458. https://doi.org/10.1074/jbc.M111.334946

Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A. / Cruys-Bagger, Nicolaj; Olsen, Jens Elmerdahl; Præstgaard, Eigil; Tatsumi, Hirosuke; Spodsberg, Nikolaj; Borch, Kim; Westh, Peter.

I: Journal of Biological Chemistry, Bind 287, Nr. 22, 2012, s. 18451-18458.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A

AU - Cruys-Bagger, Nicolaj

AU - Olsen, Jens Elmerdahl

AU - Præstgaard, Eigil

AU - Tatsumi, Hirosuke

AU - Spodsberg, Nikolaj

AU - Borch, Kim

AU - Westh, Peter

PY - 2012

Y1 - 2012

N2 - The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10–25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ∼30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.

AB - The transient kinetic behavior of enzyme reactions prior to the establishment of steady state is a major source of mechanistic information, yet this approach has not been utilized for cellulases acting on their natural substrate, insoluble cellulose. Here, we elucidate the pre-steady-state regime for the exo-acting cellulase Cel7A using amperometric biosensors and an explicit model for processive hydrolysis of cellulose. This analysis allows the identification of a pseudo-steady-state period and quantification of a processivity number as well as rate constants for the formation of a threaded enzyme complex, processive hydrolysis, and dissociation, respectively. These kinetic parameters elucidate limiting factors in the cellulolytic process. We concluded, for example, that Cel7A cleaves about four glycosidic bonds/s during processive hydrolysis. However, the results suggest that stalling the processive movement and low off-rates result in a specific activity at pseudo-steady state that is 10–25-fold lower. It follows that the dissociation of the enzyme-substrate complex (half-time of ∼30 s) is rate-limiting for the investigated system. We suggest that this approach can be useful in attempts to unveil fundamental reasons for the distinctive variability in hydrolytic activity found in different cellulase-substrate systems.

U2 - 10.1074/jbc.M111.334946

DO - 10.1074/jbc.M111.334946

M3 - Journal article

VL - 287

SP - 18451

EP - 18458

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 22

ER -

Cruys-Bagger N, Olsen JE, Præstgaard E, Tatsumi H, Spodsberg N, Borch K et al. Pre-steady-state Kinetics for Hydrolysis of Insoluble Cellulose by Cellobiohydrolase Cel7A. Journal of Biological Chemistry. 2012;287(22):18451-18458. https://doi.org/10.1074/jbc.M111.334946