LIGHT AT THE END OF A 50Å LONG TUNNEL: CRYSTAL STRUCTURES OF ENZYME-OLIGOSACCHARIDE COMPLEXES REVEAL HOW CELLOBIOHYDROLASE I BINDS CELLULOSE. Christina Divne, Jerry Ståhlberg & T. Alwyn Jones, Department of Molecular Biology, Biomedical Centre, Uppsala University, Box 590, S-751 24 Uppsala, Sweden.

We have previously reported the crystal structure of the major cellulase produced by the cellulolytic fungus Trichoderma reesei, cellobiohydrolase I (CBH I), in complex with a small saccharide ligand (1). CBH I is a retaining exo-cellulase that processively hydrolyzes alternating [[beta]]-1,4-linkages of a cellulose chain from its reducing end (1) to liberate [[beta]]-D-cellobiose as the main product. The active site is located near one end of a 50 Å long saccharide-binding tunnel and, hence, the cellulose chain has to be threaded through the tunnel prior to hydrolysis. Three carboxylate residues (E212, D214 and E217) were proposed to be involved in catalysis (1). These residues have been changed to their isosteric amide counterparts by means of site-directed mutagenesis (2) and all three mutations seriously impair the catalytic capability of the enzyme. Crystal structures of the mutant proteins in the absence of ligand have shown that no structural changes occur in their active sites (2). Crystal structures of the catalytically most deficient mutants, E212Q and E217Q, have now been successfully determined in the presence of cellotetraose, cellopentaose and cellohexaose at an effective resolution of 1.9 Å or better (3). For the first time, the complete mapping of glucose-binding sites in the tunnel of CBH I will be presented and the implications of the results for cellulose binding and catalysis will be discussed.

(1) Divne, C. et al. & Jones, A. (1994). Science 265, 524-528.

(2) Ståhlberg, J. et al. & Jones, T. A. (1996). To be published.

(3) Divne, C. et al. & Jones, T. A. (1996). To be published.