INTERACTION OF POLYSACCHARIDES WITH THE N-TERMINAL CELLULOSE-BINDING DOMAIN OF CELLULOMONAS-FIMI CENC .1. BINDING-SPECIFICITY AND CALORIMETRIC ANALYSIS
P. Tomme et al., INTERACTION OF POLYSACCHARIDES WITH THE N-TERMINAL CELLULOSE-BINDING DOMAIN OF CELLULOMONAS-FIMI CENC .1. BINDING-SPECIFICITY AND CALORIMETRIC ANALYSIS, Biochemistry, 35(44), 1996, pp. 13885-13894
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.