The kinetic theory of enzymes that modify insoluble substrates is still underdeveloped, despite the prevalence of this type of reaction both in vivo and industrial applications. Here, we present a steady-state kinetic approach to investigate inhibition occurring at the solid–liquid interface. We propose to conduct experiments under enzyme excess (E0 ≫ S0), i.e. the opposite limit compared with the conventional Michaelis–Menten framework. This inverse condition is practical for insoluble substrates and elucidates how the inhibitor reduces enzyme activity through binding to the substrate. We claim that this type of inhibition is common for interfacial enzyme reactions because substrate accessibility is low, and we show that it can be analyzed by experiments and rate equations that are analogous to the conventional approach, except that the roles of enzyme and substrate have been swapped. To illustrate the approach, we investigated the major cellulases from Trichoderma reesei (Cel6A and Cel7A) acting on insoluble cellulose. As model inhibitors, we used catalytically inactive variants of Cel6A and Cel7A. We made so-called inverse Michaelis–Menten curves at different concentrations of inhibitors and found that a new rate equation accounted well for the data. In most cases, we found a mixed type of surface-site inhibition mechanism, and this probably reflected that the inhibitor both competed with the enzyme for the productive binding-sites (competitive inhibition) and hampered the processive movement on the surface (uncompetitive inhibition). These results give new insights into the complex interplay of Cel7A and Cel6A on cellulose and the approach may be applicable to other heterogeneous enzyme reactions.
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The cover image shows high resolution of the 20 lowest energy structures of kringle 2 of human plasminogen to AGL55-NS88.2 (top) and KT155-SS1448 (bottom). To learn more about this, see the article by Qiu and colleagues (pp. 1613–1630). The image was provided by Francis J. Castellino.
Research Article|
May 29 2020
A steady-state approach for inhibition of heterogeneous enzyme reactions
Jeppe Kari
;
Jeppe Kari
*
1Department of Biotechnology and Biomedicine, Technical University of Denmark. Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark
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Corinna Schiano-di-Cola
;
Corinna Schiano-di-Cola
*
1Department of Biotechnology and Biomedicine, Technical University of Denmark. Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark
2Department of Science and Environment, Roskilde University, Universitetsvej, Building 28.C, DK-4000, Roskilde, Denmark
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Stine Fredslund Hansen;
Stine Fredslund Hansen
2Department of Science and Environment, Roskilde University, Universitetsvej, Building 28.C, DK-4000, Roskilde, Denmark
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Silke Flindt Badino
;
Silke Flindt Badino
2Department of Science and Environment, Roskilde University, Universitetsvej, Building 28.C, DK-4000, Roskilde, Denmark
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Trine Holst Sørensen
;
Trine Holst Sørensen
3Novozymes A/S, Department of Enzyme Diversity and Department of Biophysics, Biologiens Vej 2, Kgs. Lyngby, Denmark
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Ana Mafalda Cavaleiro
;
Ana Mafalda Cavaleiro
3Novozymes A/S, Department of Enzyme Diversity and Department of Biophysics, Biologiens Vej 2, Kgs. Lyngby, Denmark
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Kim Borch;
Kim Borch
3Novozymes A/S, Department of Enzyme Diversity and Department of Biophysics, Biologiens Vej 2, Kgs. Lyngby, Denmark
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Peter Westh
1Department of Biotechnology and Biomedicine, Technical University of Denmark. Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark
Correspondence: Peter Westh (petwe@dtu.dk)
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Publisher: Portland Press Ltd
Received:
January 27 2020
Revision Received:
May 07 2020
Accepted:
May 11 2020
Accepted Manuscript online:
May 11 2020
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
2020
Biochem J (2020) 477 (10): 1971–1982.
Article history
Received:
January 27 2020
Revision Received:
May 07 2020
Accepted:
May 11 2020
Accepted Manuscript online:
May 11 2020
Citation
Jeppe Kari, Corinna Schiano-di-Cola, Stine Fredslund Hansen, Silke Flindt Badino, Trine Holst Sørensen, Ana Mafalda Cavaleiro, Kim Borch, Peter Westh; A steady-state approach for inhibition of heterogeneous enzyme reactions. Biochem J 29 May 2020; 477 (10): 1971–1982. doi: https://doi.org/10.1042/BCJ20200083
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