LAUSR

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, Trp220 and 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 Trp97 is crucial in the active-site tunnel roof, resulted in a more favorable ΔS°conf relative 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.