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Trichoderma reesei cellobiohydrolase Cel6A (formerly CBHII) has a tunnel shaped active site with four internal subsites for the glucose units. We have predicted an additional ring stacking interaction for a sixth glucose moiety with a tryptophan residue (W272) found on the domain surface. Mutagenesis of this residue selectively impairs the enzyme function on crystalline cellulose but not on soluble or amorphous substrates. Our data shows that W272 forms an additional subsite at the entrance of the active site tunnel and suggests it has a specialised role in crystalline cellulose degradation, possibly in guiding a glucan chain into the tunnel.z 1998 Federation of European Biochemical Societies.

Microcystins (MCs) and anabaenopeptins from 26 cyanobacterial bloom samples dominated mainly by the genus Microcystis and collected from seven Greek freshwaters were identified and quantified by high-performance liquid chromatography coupled to a diode array detector. All the samples analyzed contained microcystins; in 27% of the samples anabaenopeptins were detected but not anabaenopeptilide (A). In each sample 1-7 microcystins and up to two anabaenopeptins (anabaenopeptins A and B) were identified. MC-RR and MC-LR were the predominant microcystins, followed by MC-YR. MC-LA and demethylated variants of MC-LR and MC-RR also were present but were not abundant. Total content of microcystin and anabaenopeptin varied from 40 to 2565 microg g(-1) freeze-dried material (mean 674.5 microg g(-1)) and from undetectable to 48 microg g(-1) freeze-dried material (mean 6.2 microg g(-1)), respectively. Qualitative and quantitative variation in the microcystins in the samples indicates there may be geographical trends in the distribution of microcystins. This study reports for the first time (1) the widespread occurrence of several different microcystins in Greek freshwaters and (2) quantitative data on the anabaenopeptins produced in natural cyanobacterial populations.

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The hydrolysis of soluble cello-oligosaccharides, with a degree of polymerisation of 4-6, catalysed by cellobiohydrolase I1 from Trichoderma reesei was studied using 'H-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-' at 27"C, are about 10-100 times faster than for the 4-methylumbelliferyl substituted analogs or for cellotriose. 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. sites of cleavage, hydrolysis stereochemistry, kinetic and binding constants as well as in in vitro mutagenesis studies [6-81. These studies have been performed mostly using 4-methylumbelliferyl p-D-glycosides derived from cellotriose, cellotetraose and cellopentaose. The binding data have been obtained by fluorescence titration techniques, which have provided association constants also for cello-oligosaccharides by means of competition experiments [9, 101. NMR spectroscopy is well suited to follow the stereochemistry of saccharides 11 I] and we have recently used it together with progress curve analysis to study the hydrolysis of cellotriose by 7: reesei CBHII [12]. Replacing the initial-rate approximation by the complete progress curve analysis was highly advantageous, since it allows the elucidation of more complex mechanisms and reduces the amount of enzyme needed .In the present work the hydrolysis of cellotetraose, cellopentame and cellohexaose by CBHII has been studied by means of proton NMR spectroscopy and HPLC while using progress curves to analyse the kinetic data.Correspondence to V. Harjunpaa, VTT Chemical Technology, P. 0.