Probing Substrate Interactions in the Active Tunnel of a Catalytically Deficient Cellobiohydrolase (Cel7)
| Autor: | Jeppe Kari, Nicolaj Cruys-Bagger, Kim Borch, Trine Holst Sørensen, Johan Pelck Olsen, Michael Skovbo Windahl, Kadri Alasepp, Peter Westh, Francieli Colussi |
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| Rok vydání: | 2015 |
| Předmět: |
Protein Conformation
Stereochemistry Oligosaccharides Calorimetry Degree of polymerization Ligands Biochemistry Catalysis Substrate Specificity Fungal Proteins Protein structure Catalytic Domain Cellulose 1 4-beta-Cellobiosidase Cellulose Molecular Biology Trichoderma reesei Trichoderma Fungal protein biology Chemistry Hydrolysis Temperature Substrate (chemistry) Cell Biology Processivity biology.organism_classification Crystallography Enzymology Regression Analysis Thermodynamics Titration Adsorption Protein Binding |
| Zdroj: | Journal of Biological Chemistry. 290:2444-2454 |
| ISSN: | 0021-9258 |
| Popis: | Cellobiohydrolases break down cellulose sequentially by sliding along the crystal surface with a single cellulose strand threaded through the catalytic tunnel of the enzyme. This so-called processive mechanism relies on a complex pattern of enzyme-substrate interactions, which need to be addressed in molecular descriptions of processivity and its driving forces. Here, we have used titration calorimetry to study interactions of cellooligosaccharides (COS) and a catalytically deficient variant (E212Q) of the enzyme Cel7A from Trichoderma reesei. This enzyme has ∼10 glucopyranose subsites in the catalytic tunnel, and using COS ligands with a degree of polymerization (DP) from 2 to 8, different regions of the tunnel could be probed. For COS ligands with a DP of 2-3 the binding constants were around 10(5) m(-1), and for longer ligands (DP 5-8) this value was ∼10(7) m(-1). Within each of these groups we did not find increased affinity as the ligands got longer and potentially filled more subsites. On the contrary, we found a small but consistent affinity loss as DP rose from 6 to 8, particularly at the higher investigated temperatures. Other thermodynamic functions (ΔH, ΔS, and ΔCp) decreased monotonously with both temperature and DP. Combined interpretation of these thermodynamic results and previously published structural data allowed assessment of an affinity profile along the length axis of the active tunnel. |
| Databáze: | OpenAIRE |
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