title: Thermodynamics of tunnel formation upon substrate binding in a processive glycoside hydrolase.
abstract: Glycoside hydrolases (GHs) catalyze the hydrolysis of glycosidic bonds and are key enzymes in carbohydrate metabolism. Efficient degradation of recalcitrant polysaccharides such as chitin and cellulose is accomplished due to synergistic enzyme cocktails consisting of accessory enzymes and mixtures of GHs with different modes of action and active site topologies. The substrate binding sites of chitinases and cellulases often have surface exposed aromatic amino acids and a tunnel or cleft topology. The active site of the exo-processive chitinase B (ChiB) from Serratia marcescens is partially closed, creating a tunnel-like catalytic cleft. To gain insight in the fundamental principles of substrate binding in this enzyme, we have studied the contribution of five key residues involved in substrate binding and tunnel formation to the thermodynamics of substrate binding. Mutation of Trp97, Phe190, Trp220and Glu221, which are all part of the tunnel walls, resulted in significant less favorable conformational entropy change (ΔS°conf) upon binding (-TΔΔS°conf= ∼5 kcal/mol). This suggest that these residues are important for the structural rigidity and pre-shaping of the tunnel prior to binding. Mutation of Asp316, which, by forming a hydrogen bond to Trp97is crucial in the active-site tunnel roof, resulted in a more favorable ΔS°confrelative to the wild type (-TΔΔS°conf= -2.2 kcal/mol). This shows that closing the tunnel-roof comes with an entropy cost, as previously suggested based on the crystal structures of GHs with tunnel topologies in complex with their substrates.
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO 5003, N-1432 Ås, Norway.
authors.forename: Anne Grethe
authors.initials: AG
authors.lastname: Hamre
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO 5003, N-1432 Ås, Norway.
authors.forename: Emil Ebbestad
authors.initials: EE
authors.lastname: Frøberg
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO 5003, N-1432 Ås, Norway.
authors.forename: Vincent G H
authors.initials: VGH
authors.lastname: Eijsink
authors.affiliation: Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, PO 5003, N-1432 Ås, Norway. Electronic address: morten.sorlie@nmbu.no.
authors.forename: Morten
authors.initials: M
authors.lastname: Sørlie
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D002688:Chitinases / Q000737:chemistry* / Q000235:genetics; D019277:Entropy; D008958:Models, Molecular; D020125:Mutation, Missense; D012706:Serratia marcescens / Q000201:enzymology*
chemical_list: C482346:ChiB protein, Serratia marcescens; D002688:Chitinases