Specificity and Affinity of Substrate Binding by a Family 17 Carbohydrate-Binding Module from Clostridium cellulovorans Cellulase 5A

Abstract

The C-terminal carbohydrate-binding module (CBM17) from Clostridium cellulovorans cellulase 5A is a β-1,4-glucan binding module with a preference for soluble chains. CBM17 binds to phosphoric acid swollen Avicel (PASA) and Avicel with association constants of 2.9 (±0.2) × 10⁵ and 1.6 (±0.2) × 10⁵ M^-1, respectively. The capacity values for PASA and Avicel were 11.9 and 0.4 μmol/g of cellulose, respectively. CBM17 did not bind to crystalline cellulose. CBM17 bound tightly to soluble barley β-glucan and the derivatized celluloses HEC, EHEC, and CMC. The association constants for binding to barley β-glucan, HEC, and EHEC were approximately 2.0 × 10⁵ M^-1. Significant binding affinities were found for cello-oligosaccharides greater than three glucose units in length. The affinities for cellotriose, cellotetraose, cellopentaose, and cellohexaose were 1.2 (±0.3) × 10³, 4.3 (±0.4) × 10³, 3.8 (±0.1) × 10⁴, and 1.5 (±0.0) × 10⁵ M^-1, respectively. Fluorescence quenching studies and N-bromosuccinimide modification indicate the participation of tryptophan residues in ligand binding. The possible architecture of the ligand-binding site is discussed in terms of its binding specificity, affinity, and the participation of tryptophan residues.