title: Free Energy Diagram for the Heterogeneous Enzymatic Hydrolysis of Glycosidic Bonds in Cellulose.
abstract: Kinetic and thermodynamic data have been analyzed according to transition state theory and a simplified reaction scheme for the enzymatic hydrolysis of insoluble cellulose. For the cellobiohydrolase Cel7A from Hypocrea jecorina (Trichoderma reesei), we were able to measure or collect relevant values for all stable and activated complexes defined by the reaction scheme and hence propose a free energy diagram for the full heterogeneous process. For other Cel7A enzymes, including variants with and without carbohydrate binding module (CBM), we obtained activation parameters for the association and dissociation of the enzyme-substrate complex. The results showed that the kinetics of enzyme-substrate association (i.e. formation of the Michaelis complex) was almost entirely entropy-controlled and that the activation entropy corresponded approximately to the loss of translational and rotational degrees of freedom of the dissolved enzyme. This implied that the transition state occurred early in the path where the enzyme has lost these degrees of freedom but not yet established extensive contact interactions in the binding tunnel. For dissociation, a similar analysis suggested that the transition state was late in the path where most enzyme-substrate contacts were broken. Activation enthalpies revealed that the rate of dissociation was far more temperature-sensitive than the rates of both association and the inner catalytic cycle. Comparisons of one- and two-domain variants showed that the CBM had no influence on the transition state for association but increased the free energy barrier for dissociation. Hence, the CBM appeared to promote the stability of the complex by delaying dissociation rather than accelerating association.
authors.affiliation: From Roskilde University, NSM, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Roskilde, Denmark and.
authors.forename: Trine Holst
authors.initials: TH
authors.lastname: Sørensen
authors.affiliation: From Roskilde University, NSM, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Roskilde, Denmark and.
authors.forename: Nicolaj
authors.initials: N
authors.lastname: Cruys-Bagger
authors.affiliation: Novozymes A/S, Krogshøjvej 36, DK-2880 Bagsværd, Denmark.
authors.forename: Kim
authors.initials: K
authors.lastname: Borch
authors.affiliation: From Roskilde University, NSM, Research Unit for Functional Biomaterials, 1 Universitetsvej, Building 28, DK-4000 Roskilde, Denmark and pwesth@ruc.dk.
authors.forename: Peter
authors.initials: P
authors.lastname: Westh
mesh_terms: D000465:Algorithms; D055162:Biocatalysis; D002482:Cellulose / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000235:genetics / Q000378:metabolism*; D019277:Entropy; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D006868:Hydrolysis; D035901:Hypocrea / Q000201:enzymology / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011485:Protein Binding; D013379:Substrate Specificity; D013816:Thermodynamics; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics
chemical_list: D005656:Fungal Proteins; D007527:Isoenzymes; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: Hypocrea jecorina; Rasamsonia emersonii; carbohydrate-binding protein; cellulase; enzyme kinetics; enzyme mechanism; thermodynamics