title: Probing substrate interactions in the active tunnel of a catalytically deficient cellobiohydrolase (Cel7).
abstract: 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.
authors.affiliation: From the Roskilde University, NSM, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Denmark and.
authors.forename: Francieli
authors.initials: F
authors.lastname: Colussi
authors.affiliation: From the Roskilde University, NSM, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Denmark and.
authors.forename: Trine H
authors.initials: TH
authors.lastname: Sørensen
authors.affiliation: From the Roskilde University, NSM, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Denmark and.
authors.forename: Kadri
authors.initials: K
authors.lastname: Alasepp
authors.affiliation: From the Roskilde University, NSM, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Denmark and.
authors.forename: Jeppe
authors.initials: J
authors.lastname: Kari
authors.affiliation: From the Roskilde University, NSM, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Denmark and.
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Cruys-Bagger
authors.affiliation: From the Roskilde University, NSM, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Denmark and Novozymes A/S, Krogshøjvej 36, DK-2880, Bagsværd, Denmark.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Windahl
authors.affiliation: From the Roskilde University, NSM, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Denmark and.
authors.forename: Johan P
authors.initials: JP
authors.lastname: Olsen
authors.affiliation: Novozymes A/S, Krogshøjvej 36, DK-2880, Bagsværd, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.affiliation: From the Roskilde University, NSM, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Denmark and pwesth@ruc.dk.
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D000327:Adsorption; D002151:Calorimetry; D002384:Catalysis; D020134:Catalytic Domain; D002482:Cellulose / Q000737:chemistry*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry; D006868:Hydrolysis; D008024:Ligands; D009844:Oligosaccharides / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D012044:Regression Analysis; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D014242:Trichoderma / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D008024:Ligands; D009844:Oligosaccharides; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: Cellulase; Enzyme Mechanism; Glycoside Hydrolase; Isothermal Titration Calorimetry (ITC); Processivity; Protein Engineering; Thermodynamics