STRUCTURAL STUDIES ON CELLULASE-OLIGOSACCHARIDE COMPLEXES Gideon Davies1, Gerlind Sulzenbacher1, Bernard Henrissat2, Hugues Driguez2 and Martin Schülein3. 1.Department of Chemistry, University of York, Heslington, York, Y01 5AT, Great Britain, 2. CERMAV, CNRS, BP-53, F38041, Grenoble, France and 3. Novo-Nordisk a/s, Novo allé, 2880-Bagsvaerd, Denmark.
Since the seminal structure determination of hen-egg white lysozyme over 25 years ago, the number of glycosyl hydrolase structures has blossomed. There are now over 54 sequenced-based families of glycosyl hydrolases with structural representatives for approximately 25 of these. Oligosaccharide-bound structures are somewhat more scarce. We have been studying the cellulases, enzymes which hydrolyse the [[beta]]-1,4 linkages of cellulose. In particular, the endoglucanase V from Humicola insolens, which performs catalysis with inversion of the anomeric configuration and the endoglucanase I's from H. insolens and Fusarium oxysporum which both act with a net retention of configuration.
We have obtained saccharide-bound complexes of these inverting and retaining endoglucanases by techniques including:
* mechanism-based inhibitors and suicide substrates
* active -enzyme with reaction products
* inactive-mutants plus substrates
* non-hydrolysable substrate analogues
Analysis of these structures reveals the methods utilised by these enzymes to facilitate catalysis, such as substrate distortion and favoured binding of the elongated glycosidic bond found in the transition state of the glycosyl hydrolysis. The non-hydrolysable substrate analogue is particularly interesting since it provides the first glimpse of an intact oligosaccharide bound across the active site of an enzyme. The pyranaose ring is distorted into a boat conformation which gives a pseudo-axial conformation for the glycosidic bond and leaving group as predicted by David Phillips in 1974 and as expected on stereoelectronic grounds.