Cello-oligosaccharide hydrolysis by cellobiohydrolase II from Trichoderma reesei. Association and rate constants derived from an analysis of progress curves

The hydrolysis of soluble cello-oligosaccharides, with a degree of polymerisation of 4-6, catalysed by cellobiohydrolase II from Trichoderma reesei was studied using 1H-NMR spectroscopy and HPLC. The experimental progress curves were analysed by fitting numerically integrated kinetic equations, which provided cleavage patterns and kinetic constants for each oligosaccharide. This analysis procedure accounts for product inhibition and avoids the initial slope approximation. No glucose was detected at the beginning of the reaction indicating that only the internal glycosidic linkages are attacked. For cellotetraose only the second glycosidic linkage was cleaved. For cellopentaose and cellohexaose the second and the third glycosidic linkage from the non-reducing end were cleaved with approximately equal probability. The degradation rates of these cello-oligosaccharides, 1-12 s-1 at 27 degrees C, are about 10-100 times faster than for the 4-methylumbelliferyl substituted analogs or for collotriose. No intermediate products larger than cellotriose were released. The degradation rate for cellotetraose were higher than its off-rate, which accounts for the processive degradation of cellohexaose. A high cellohexaose/enzyme ratio caused slow reversible inactivation of the enzyme.