Rate of Threading a Cellulose Chain into the Binding Tunnel of a Cellulase

Nicolaj Cruys-Bagger, Kadri Alasepp, Morten Andersen, Johnny T. Ottesen, Kim Borch, Peter Westh

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Resumé

Industrially important cellulase Cel7A hydrolyzes crystalline cellulose by a complex processive mechanism in which the enzyme slides along the cellulose surface with one strand of the polymeric substrate channeled through its catalytic tunnel. Each processive run must start with threading the tunnel with a cellulose strand and end with the opposite, that is, the dethreading process. Evidence has suggested that threading or dethreading may be rate-limiting for the overall enzyme reaction. To directly elucidate the rates of threading and dethreading, we analyzed experimental data with respect to a two-step model that distinguishes enzymes in free, associated nonthreaded, and threaded states. This approach enabled the estimation of rate constants for both steps in both directions. The results showed that Cel7A utilizes a “tapping” mode of attack, in which it associates unproductively with the cellulose surface many times before it eventually finds a location at which it gets threaded. Moreover, it was concluded that at the quasi steady state dethreading was the main determinant of the overall hydrolytic rate under most conditions. An exception to this was at very low enzyme/substrate ratios, at which other steps also influenced the overall dynamics. These results will be helpful in identifying rate-limiting steps for cellulases and, in turn, targets for rational design of faster enzymes.
OriginalsprogEngelsk
TidsskriftThe Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical
Vol/bind120
Udgave nummer25
Antal sider10
ISSN1520-6106
DOI
StatusUdgivet - 2016

Citer dette

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title = "Rate of Threading a Cellulose Chain into the Binding Tunnel of a Cellulase",
abstract = "Industrially important cellulase Cel7A hydrolyzes crystalline cellulose by a complex processive mechanism in which the enzyme slides along the cellulose surface with one strand of the polymeric substrate channeled through its catalytic tunnel. Each processive run must start with threading the tunnel with a cellulose strand and end with the opposite, that is, the dethreading process. Evidence has suggested that threading or dethreading may be rate-limiting for the overall enzyme reaction. To directly elucidate the rates of threading and dethreading, we analyzed experimental data with respect to a two-step model that distinguishes enzymes in free, associated nonthreaded, and threaded states. This approach enabled the estimation of rate constants for both steps in both directions. The results showed that Cel7A utilizes a “tapping” mode of attack, in which it associates unproductively with the cellulose surface many times before it eventually finds a location at which it gets threaded. Moreover, it was concluded that at the quasi steady state dethreading was the main determinant of the overall hydrolytic rate under most conditions. An exception to this was at very low enzyme/substrate ratios, at which other steps also influenced the overall dynamics. These results will be helpful in identifying rate-limiting steps for cellulases and, in turn, targets for rational design of faster enzymes.",
author = "Nicolaj Cruys-Bagger and Kadri Alasepp and Morten Andersen and Ottesen, {Johnny T.} and Kim Borch and Peter Westh",
year = "2016",
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journal = "Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical",
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Rate of Threading a Cellulose Chain into the Binding Tunnel of a Cellulase. / Cruys-Bagger, Nicolaj; Alasepp, Kadri; Andersen, Morten; Ottesen, Johnny T.; Borch, Kim; Westh, Peter.

I: The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical, Bind 120, Nr. 25, 2016.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningpeer review

TY - JOUR

T1 - Rate of Threading a Cellulose Chain into the Binding Tunnel of a Cellulase

AU - Cruys-Bagger, Nicolaj

AU - Alasepp, Kadri

AU - Andersen, Morten

AU - Ottesen, Johnny T.

AU - Borch, Kim

AU - Westh, Peter

PY - 2016

Y1 - 2016

N2 - Industrially important cellulase Cel7A hydrolyzes crystalline cellulose by a complex processive mechanism in which the enzyme slides along the cellulose surface with one strand of the polymeric substrate channeled through its catalytic tunnel. Each processive run must start with threading the tunnel with a cellulose strand and end with the opposite, that is, the dethreading process. Evidence has suggested that threading or dethreading may be rate-limiting for the overall enzyme reaction. To directly elucidate the rates of threading and dethreading, we analyzed experimental data with respect to a two-step model that distinguishes enzymes in free, associated nonthreaded, and threaded states. This approach enabled the estimation of rate constants for both steps in both directions. The results showed that Cel7A utilizes a “tapping” mode of attack, in which it associates unproductively with the cellulose surface many times before it eventually finds a location at which it gets threaded. Moreover, it was concluded that at the quasi steady state dethreading was the main determinant of the overall hydrolytic rate under most conditions. An exception to this was at very low enzyme/substrate ratios, at which other steps also influenced the overall dynamics. These results will be helpful in identifying rate-limiting steps for cellulases and, in turn, targets for rational design of faster enzymes.

AB - Industrially important cellulase Cel7A hydrolyzes crystalline cellulose by a complex processive mechanism in which the enzyme slides along the cellulose surface with one strand of the polymeric substrate channeled through its catalytic tunnel. Each processive run must start with threading the tunnel with a cellulose strand and end with the opposite, that is, the dethreading process. Evidence has suggested that threading or dethreading may be rate-limiting for the overall enzyme reaction. To directly elucidate the rates of threading and dethreading, we analyzed experimental data with respect to a two-step model that distinguishes enzymes in free, associated nonthreaded, and threaded states. This approach enabled the estimation of rate constants for both steps in both directions. The results showed that Cel7A utilizes a “tapping” mode of attack, in which it associates unproductively with the cellulose surface many times before it eventually finds a location at which it gets threaded. Moreover, it was concluded that at the quasi steady state dethreading was the main determinant of the overall hydrolytic rate under most conditions. An exception to this was at very low enzyme/substrate ratios, at which other steps also influenced the overall dynamics. These results will be helpful in identifying rate-limiting steps for cellulases and, in turn, targets for rational design of faster enzymes.

U2 - 10.1021/acs.jpcb.6b01877

DO - 10.1021/acs.jpcb.6b01877

M3 - Journal article

VL - 120

JO - Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical

JF - Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical

SN - 1520-6106

IS - 25

ER -