@article { ISI:000079646300001,
	title = {1998 Hoffmann la Roche Award Lecture - Understanding and exploiting glycosidases},
	journal = {CANADIAN JOURNAL OF CHEMISTRY-REVUE CANADIENNE DE CHIMIE},
	volume = {77},
	number = {1},
	year = {1999},
	note = {81st Canadian-Society-for-Chemistry Conference, WHISTLER, CANADA, JUN 01-02, 1998},
	month = {JAN},
	pages = {1-11},
	publisher = {Canadian Soc Chem},
	abstract = {Glycosidases fall into two major mechanistic classes; those that hydrolyse the glycosidic bond with retention of anomeric configuration and those that do so with inversion. Retaining glycosidases employ a mechanism involving a covalent glycosyl-enzyme intermediate formed and hydrolysed with acid-base catalytic assistance via oxocarbenium ion-like transition states. This intermediate has been trapped in two distinct ways, either by modification of the substrate through fluorination, or of the enzyme through mutation of key residues. This allows the amino acid residue to which this sugar is attached to be identified through LC/MS-MS analysis of peptic digests. Three-dimensional structures of several of these glycosyl-enzyme complexes, along with those of Michaelis complexes, have been determined through X-ray crystallographic analysis, revealing the identities of important amino acid residues involved in catalysis, particularly the involvement of the catalytic nucleophile in strong hydrogen bonding to the sugar 2-hydroxyl. They have also revealed evidence of substantial substrate distortion upon binding. Insight into the function of the acid-base catalyst Glu172 in Bacillus circulans xylanase has been obtained through NMR titration of side chain C-13-labelled glutamic acid enzyme both free and in the 2-fluoroxylobiosyl-enzyme complex. The pK(a) of Glu172 is relatively high in the free enzyme, consistent with its role as an acid catalyst, but drops 2.5 units upon formation of the intermediate, consistent with its new role as a base catalyst. This {\textquoteleft}{\textquoteleft}cycling{{\textquoteright}{\textquoteright}} of the pK(a) is shown to be a direct consequence of the change in charge of the nucleophile, Glu78. Finally, an efficient catalyst for synthesis, but not hydrolysis, of glycosidic bonds has been generated by mutation of the glutamic acid catalytic nucleophile of a beta-glucosidase to an alanine. When used with alpha-glucosyl fluoride as a glycosyl donor, along with a suitable acceptor, oligosaccharides up to five sugars in length have been made with yields of up to 90\% on individual steps. These new enzymes have been named glycosynthases.},
	issn = {0008-4042},
	doi = {10.1139/cjc-77-1-1},
	author = {Withers, SG}
}
