Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose

Leigh Murphy, Nicolaj Cruys-Bagger, Martin J. Baumann, Søren Nymand Olsen, Kim Borch, Søren Flensted Lassen, Matthew D. Sweeney, Hirosuke Tatsumi, Peter Westh

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

Resumé

The kinetics of cellulose hydrolysis have longbeen described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies.Wehave used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s -1 at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.
OriginalsprogEngelsk
TidsskriftJournal of Biological Chemistry
Vol/bind287
Udgave nummer2
Sider (fra-til)1252-1260
ISSN0021-9258
StatusUdgivet - 6 jan. 2012

Citer dette

Murphy, L., Cruys-Bagger, N., Baumann, M. J., Olsen, S. N., Borch, K., Lassen, S. F., ... Westh, P. (2012). Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose. Journal of Biological Chemistry, 287(2), 1252-1260.
Murphy, Leigh ; Cruys-Bagger, Nicolaj ; Baumann, Martin J. ; Olsen, Søren Nymand ; Borch, Kim ; Lassen, Søren Flensted ; Sweeney, Matthew D. ; Tatsumi, Hirosuke ; Westh, Peter. / Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose. I: Journal of Biological Chemistry. 2012 ; Bind 287, Nr. 2. s. 1252-1260.
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abstract = "The kinetics of cellulose hydrolysis have longbeen described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies.Wehave used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s -1 at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.",
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Murphy, L, Cruys-Bagger, N, Baumann, MJ, Olsen, SN, Borch, K, Lassen, SF, Sweeney, MD, Tatsumi, H & Westh, P 2012, 'Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose', Journal of Biological Chemistry, bind 287, nr. 2, s. 1252-1260.

Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose. / Murphy, Leigh; Cruys-Bagger, Nicolaj; Baumann, Martin J.; Olsen, Søren Nymand; Borch, Kim ; Lassen, Søren Flensted; Sweeney, Matthew D.; Tatsumi, Hirosuke; Westh, Peter.

I: Journal of Biological Chemistry, Bind 287, Nr. 2, 06.01.2012, s. 1252-1260.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose

AU - Murphy, Leigh

AU - Cruys-Bagger, Nicolaj

AU - Baumann, Martin J.

AU - Olsen, Søren Nymand

AU - Borch, Kim

AU - Lassen, Søren Flensted

AU - Sweeney, Matthew D.

AU - Tatsumi, Hirosuke

AU - Westh, Peter

PY - 2012/1/6

Y1 - 2012/1/6

N2 - The kinetics of cellulose hydrolysis have longbeen described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies.Wehave used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s -1 at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.

AB - The kinetics of cellulose hydrolysis have longbeen described by an initial fast hydrolysis rate, tapering rapidly off, leading to a process that takes days rather than hours to complete. This behavior has been mainly attributed to the action of cellobiohydrolases and often linked to the processive mechanism of this exo-acting group of enzymes. The initial kinetics of endo-glucanases (EGs) is far less investigated, partly due to a limited availability of quantitative assay technologies.Wehave used isothermal calorimetry to monitor the early time course of the hydrolysis of insoluble cellulose by the three main EGs from Trichoderma reesei (Tr): TrCel7B (formerly EG I), TrCel5A (EG II), and TrCel12A (EG III). These endo-glucanases show a distinctive initial burst with a maximal rate that is about 5-fold higher than the rate after 5 min of hydrolysis. The burst is particularly conspicuous for TrCel7B, which reaches a maximal turnover of about 20 s -1 at 30 °C and conducts about 1200 catalytic cycles per enzyme molecule in the initial fast phase. For TrCel5A and TrCel12A the extent of the burst is 2-300 cycles per enzyme molecule. The availability of continuous data on EG activity allows an analysis of the mechanisms underlying the initial kinetics, and it is suggested that the slowdown is linked to transient inactivation of enzyme on the cellulose surface. We propose, therefore, that the frequency of structures on the substrate surface that cause transient inactivation determine the extent of the burst phase.

M3 - Journal article

VL - 287

SP - 1252

EP - 1260

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 2

ER -

Murphy L, Cruys-Bagger N, Baumann MJ, Olsen SN, Borch K, Lassen SF et al. Origin of initial burst in activity for Trichoderma reesei endo-glucanases hydrolyzing insoluble cellulose. Journal of Biological Chemistry. 2012 jan 6;287(2):1252-1260.