Specificity and affinity of substrate binding by a family 17 carbohydrate-binding module from Clostridium cellulovorans cellulase 5A

The C-terminal carbohydrate-binding module (CBM17) from Clostridium cellulo vorans cellulase 5A is a beta-1,4-glucan binding module with a preference f or soluble chains. CBM17 binds to phosphoric acid swollen Avicel (PASA) and Avicel with association constants of 2.9 (+/-0.2) x 10(5) and 1.6 (+/-0.2) x 10(5) M-1, respectively. The capacity values for PASA and Avicel were 11 .9 and 0.4 mu mol/g of cellulose, respectively. CBM17 did not bind to cryst alline cellulose. CBM17 bound tightly to soluble barley beta-glucan and the derivatized celluloses HEC, EHEC, and CMC. The association constants for b inding to barley beta-glucan, HEC, and EHEC were approximately 2.0 x 105 M- 1. Significant binding affinities were found for cello-oligosaccharides gre ater than three glucose units in length. The affinities for cellotriose, ce llotetraose, cellopentaose, and cellohexaose were 1.2 (+/-0.3) x 10(3), 4.3 (+/-0.4) x 10(3), 3.8 (+/-0.1) x 10(4), and 1.5 (+/-0.0) x 10(5) M-1, resp ectively. Fluorescence quenching studies and N-bromosuccinimide modificatio n indicate the participation of tryptophan residues in ligand binding. The possible architecture of the ligand-binding site is discussed in terms of i ts binding specificity, affinity, and the participation of tryptophan resid ues.