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Limited action of papain on the native forms of two cellobiohydrolases (CBH) from Trichoderma reesei (CBH I, 65 kDa, and CBH II,58 kDa) leads to the isolation of the respective core fragments (56 kDa and 45 kDa) which are fully active on small, soluble substrates, but have a strongly reduced activity (respectively 10% and 50% of the initial value) on microcrystalline cellulose (Avicel).By partial sequencing at the C terminus of the CBH I core and at the N terminus of the CBH I1 core the papain cleavage sites have been assigned in the primary structures (at about residue 431 and 82 respectively). This limited action of papain on the native enzymes indicates the presence of hinge regions linking the core to these terminal glycopeptides. The latter conserved sequences appear either at the C or N terminus of several cellulolytic enzymes from ~r~c~o d e r m a reesei [Teeri et al. (1987) Gene 51,43 -521. The specific activities of the intact enzymes and their cores on two forms of insoluble cellulose (crystalline, amorphous) differentiate the CBH I and CBH TI in terms of adsorption and catalytic properties, Distinct functions can be attributed to the terminal peptides: for intact CBH I1 the N-terminal region contributes in the binding onto both cellulose types; the homologous C-terminal peptide in CBH I, however, only affects the interaction with microcrystalline cellulose. It could be inferred that CBH I and its core bind preferentially to crystalline regions. This seems to be corroborated by the results of CBH I/CBH I1 synergism experiments.The filamentous fungus Trichoderma reesei presents an ideal system for the study of cellulose hydrolysis since it produces a variety of enzymes including endocellulase and exocellulase (cellobiohydrolases) and b-glucosidases (cellobiases) [l]. The primary structure of several of these proteins is presently known [2 -61 but no structure/function relationships have yet been established. The two cellobiohydrolases, CBH I and CBH I1 [7 -111, lack any apparent homology in their amino acid sequence except for a conserved region present at their C and N termini respectively [4].Their action is similar both with regard to their strong adsorption onto insoluble substrates and lack of carboxymethylcellulase activity. However, studies both with microcrystalline cellulose [9] and small, soluble substrates [I 21 reveal some differences in specificity. In addition, the two cellobiohydrolases interact synergistically both with each other and with endoglucanases on microcrystalline substrates In order to differentiate these enzymes further by their function and to investigate possible domain structure, proteolysis studies were performed. Preliminary results on the limited action of papain on native CBH I have been reported by us [16]. Two fragments were isolated: first, a heavily glycosylated peptide, tentatively identified with the C terminus; secondly, a core protein containing the active (hydrolytic) site. The terminal peptide was characterised as an independent structural and functional dom...

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Limited proteolysis of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei by papain yields a core protein (56 kDa) which is fully active against small, soluble substrates such as the chromophoric glycosides derived from the cellodextrins and lactose. Activity against an insoluble substrate, such as Avicel, is however completely lost and concomitantly decreased adsorption onto this microcrystalline cellulose is observed. The peptide (10 kDa), initially split off during proteolysis, is identified as the heavily glycosylated carboxy‐terminal of the native CBH I. Depending on the experimental conditions the core protein is further nicked in between disulfide bonds, but its properties and stability do not appreciably differ from those of intact CBH I. These results lead to the proposal of a bifunctional organisation of the CBH I: one domain, corresponding to the carboxyterminal, acts as a binding site for insoluble cellulose and the other, localised in the core protein, contains the active (hydrolytic) site.

The carbohydrate-binding specificity of the N-terminal cellulose-binding domain (CBDN1) from Cellulomonas fimi beta-1,4-glucanase C (CenC) was investigated using affinity electrophoresis, binding assays and microcalorimetry in parallel with NMR and difference ultraviolet absorbance spectroscopy [Johnson, P.E., Tomme, P., Joshi, M.D., & McIntosh, I., P. (1996) Biochemistry 35, 13895-13906]. Binding of CBDN1 on insoluble cellulose is distinctly different from other cellulose-binding domains. CBDN1 binds amorphous cellulose (phosphoric acid-swollen) with high affinity (Kr = 5.1 L g-1), binds Avicel weakly and does not bind highly crystalline bacterial or tunicin cellulose. Moreover, CBDN1 binds soluble cellooligosaccharides and beta-1,4-linked oligomers of glucose such as hydroxyethycellulose, soluble beta-1,3-1,4-glucans from barley and oat, but has no affinity for alpha-1,4-, beta-1,3-, or beta-1,6-polymers of glucose. This is the first report of a cellulose-binding domain with strong and specific affinity for soluble glycans. The thermodynamics for binding of CBDN1 to oligosaccharides, soluble glycans, and phosphoric acid-swollen cellulose were investigated by titration microcalorimetry. At least four beta-1,4-linked glucopyranosides are required to detect binding. For larger glucans, with five or more glucopyranoside units, the binding constants and standard free energy changes are virtually independent of the glucan chain length, indicating that cellopentaose completely fills the binding site. Binding is moderately strong with binding constants ranging from 3,200 +/- 500 M-1 for cellotetraose, to 25,000 +/- 3,000 M-1 for the larger sugars. The reactions are controlled by favorable standard free enthalpy changes which are compensated in a linear fashion by a significant decrease in entropy. A predominance of polar interactions such as hydrogen bonding together with van der Waals interactions provide the major driving forces for the binding event.