Studies of the cellulolytic system of Trichoderma reesei QM 9414

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Abstract
Binding onto cellobiohydrolase II from Trichoderma reesei of glucose, cellobiose, cellotriose, derivatized and analogous compounds, is monitored by protein-difference-absorption spectroscopy and by titration of ligand fluorescence, either at equilibrium or by the stopped-flow technique. The data complete earlier results [van Tilbeurgh, H., Pettersson, L. G., Bhikhabhai, R., De Boeck, H. and Claeyssens, M. (1985) Eur. J. Biochem. 148, 329–334] indicating an extended active center, with putative subsites ABCD. Subsite A specifically complexes with β-d-glucosides and d-glucose; at 25°C the latter influences the concomitant binding of other ligands at neighbouring sites. For several ligands this cooperative effect for binding (at 0.33 M glucose and temperature range 4–37°C) was characterized by a substantial increase of the enthalpic term (ΔΔH°=–35 kJ mol–1). Glucose (0.33 M) decreases the association and dissociation rate parameters of 4-methylumbelliferyl β-d-cellobioside by one order of magnitude: k+= (3.6 ± 0.5)×10–5 M–1 s–1 versus (5.1 ± 0.1)×10–6 M–1 s–1 (in the absence of glucose) and k−= (1.3 ± 0.1) s−1 versus (14.0 ± 0.3) s–1. As deduced from substrate-specificity studies and inhibition experiments, subsite B interacts with terminal non-reducing glucopyranosyl residues of oligomeric ligands and substrates, whereas catalytic (hydrolytic) cleavage occurs between C and D. Association constants 10–100 times higher than those for cellobiose or its glycosides were observed for d-glucopyranosyl-(1 → 4)-β-d-xylopyranose and cellobionolactone derivatives, suggesting ‘transition-state’-type binding for these ligands at subsite C. Although subsite D can accomodate a bulky chromophoric group (MeUmb) its preference for a glucosyl residue is reflected in the lower binding enthalpy of cellotriose (–34 kJ mol–1) as compared to cellobiose (–28.3 kJ mol–1) and MeUmb(Glc)2 (–11.6 kJ mol–1). This model indicates that oligomeric ligands (substrates) interact through cooperativity of their subunits at the extended binding site of cellobiohydrolase II.