Enzyme Kinetics by GH7 Cellobiohydrolases on Chromogenic Substrates is Dictated by Non-Productive Binding: Insights from Crystal Structures and MD Simulation

Topi Haataja, Japheth Gado, Anu Nutt, Nolan Anderson, Mikael Nilsson, Majid Momeni, Roland Isaksson, Priit Valjamae, Gunnar Johansson, Christina Payne, Jerry Stahlberg

Research output: Contribution to journalArticlepeer-review

3 Scopus Citations


Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) (EC3.2.1.176) are the major cellulose degrading enzymes both in industrial settings and in the context of carbon cycling in nature. Small carbohydrate conjugates such as p-nitrophenyl-β-d-cellobioside (pNPC), p-nitrophenyl-β-d-lactoside (pNPL) and methylumbelliferyl-β-d-cellobioside have commonly been used in colorimetric and fluorometric assays for analysing activity of these enzymes. Despite the similar nature of these compounds the kinetics of their enzymatic hydrolysis vary greatly between the different compounds as well as among different enzymes within the GH7 family. Through enzyme kinetics, crystallographic structure determination, molecular dynamics simulations, and fluorometric binding studies using the closely related compound o-nitrophenyl-β-d-cellobioside (oNPC), in this work we examine the different hydrolysis characteristics of these compounds on two model enzymes of this class, TrCel7A from Trichoderma reesei and PcCel7D from Phanerochaete chrysosporium. Protein crystal structures of the E212Q mutant of TrCel7A with pNPC and pNPL, and the wildtype TrCel7A with oNPC, reveal that non-productive binding at the product site is the dominating binding mode for these compounds. Enzyme kinetics results suggest the strength of non-productive binding is a key determinant for the activity characteristics on these substrates, with PcCel7D consistently showing higher turnover rates (kcat) than TrCel7A, but higher Michaelis–Menten (KM) constants as well. Furthermore, oNPC turned out to be useful as an active-site probe for fluorometric determination of the dissociation constant for cellobiose on TrCel7A but could not be utilized for the same purpose on PcCel7D, likely due to strong binding to an unknown site outside the active site.

Original languageAmerican English
Pages (from-to)379-399
Number of pages21
JournalFEBS Journal
Issue number2
StatePublished - 2023

Bibliographical note

Publisher Copyright:
© 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

NREL Publication Number

  • NREL/JA-2800-84058


  • Cel7
  • cellulase
  • fluorescence
  • ligand binding
  • Phanerochaete chrysosporium
  • Trichoderma reesei


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