INTERACTION OF POLYSACCHARIDES WITH THE N-TERMINAL CELLULOSE-BINDING DOMAIN OF CELLULOMONAS-FIMI CENC .1. BINDING-SPECIFICITY AND CALORIMETRIC ANALYSIS

The carbohydrate-binding specificity of the N-terminal cellulose-bindi ng domain (CBDN1) from Cellulomonas fimi beta-1,4-glucanase C (CenC) w as investigated using affinity electrophoresis, binding assays and mic rocalorimetry in parallel with NMR and difference ultraviolet absorban ce spectroscopy [Johnson, P. E., Tomme, P., Joshi, M. D., & McIntosh, L. P. (1996) Biochemistry 35, 13895-13906]. Binding of CBDN1 on insolu ble cellulose is distinctly different from other cellulose-binding dom ains. CBDN1 binds amorphous cellulose (phosphoric acid-swollen) with h igh affinity (K-f = 5.1 L g(-1)), binds Avicel weakly and does not bin d highly crystalline bacterial or tunicin cellulose. Moreover, CBDN1 b inds soluble cellooligosaccharides and beta-1,4-linked oligomers of gl ucose such as hydroxyethylcellulose, soluble beta-1,3-1,4-glucans from barley and oat, but has no affinity for alpha-1,4-, beta-1,3-, or bet a-1,6-polymers of glucose. This is the first report of a cellulose-bin ding domain with strong and specific affinity for soluble glycans. The thermodynamics for binding of CBDN1 to oligosaccharides, soluble glyc ans, and phosphoric acid-swollen cellulose were investigated by titrat ion microcalorimetry. At least four beta-1,4-linked glucopyranosides a re required to detect binding. For larger glucans, with five or more g lucopyranoside units, the binding constants and standard free energy c hanges are virtually independent of the glucan chain length, indicatin g that cellopentaose completely fills the binding site. Binding is mod erately strong with binding constants ranging from 3 200 +/- 500 M(-1) for cellotetraose, to 25 000 +/- 3 000 M(-1) for the larger sugars. T he reactions are controlled by favorable standard free enthalpy change s which are compensated in a linear fashion by a significant decrease in entropy. A predominance of polar interactions such as hydrogen bond ing together with van der Waals interactions provide the major driving forces for the binding event.