1998 Hoffmann La Roche Award Lecture Understanding and exploiting glycosidases

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 13 C-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 "cycling" 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 β-glucosidase to an alanine. When used with α-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.Résumé : Les glycosidases appartiennent à deux grandes classes de mécanismes : celles qui hydrolysent la liaison glycosidique avec rétention de la configuration anomérique et celles qui le font avec inversion. Les glycosidases qui conduisent à une rétention font appel à un mécanisme avec formation d'un intermédiaire covalent glycosyl-enzyme et une hydrolyse assistée par une catalyse acide-base comportant un état de transition qui ressemble à un ion oxocarbénium. On a piégé cet intermédiaire de deux façons distinctes en procédant à une modification du substrat par fluoration ou de l'enzyme par mutation de résidus clés. Une analyse par chromatographie liquide/spectrométrie de masse -spectrométrie de masse (CL/SM-SM) sur les produits de digestion peptique permet alors d'identifier l'acide aminé auquel le sucre est attaché. Faisant appel aux méthodes d'analyse cristallographiques par diffraction des rayons X, on a déterminé les structures trid...

Selectfluor: Mechanismen und Anwendungen

“…Den Ergebnissen zufolge könnten sich die Mechanismen, mit denen die beiden Galactosyltransferasen die Glycosylierung katalysieren, stark unterscheiden. Ein ähnlich unterschiedliches Verhalten wurde bei konfigurationsinvertierenden und ‐erhaltenden Glycosidasen beschrieben 107. Für die konfigurationserhaltende α‐1,3‐GalT wurde als Mechanismus eine doppelte Substitution angenommen, und es wurde untersucht, ob 19 das Enzym langsam inaktiviert.…”

Nature's many mechanisms for the degradation of oligosaccharides

“…13 This class of enzymes has been the subject of extensive studies, and the interested reader is referred to a number of excellent recent reviews. 1,[14][15][16][17][18][19][20][21][22] Glycoside hydrolases are classified into different families based on sequence similarity, and this information can be found on the frequently updated CAZY Web site (http://afmb.cnrsmrs.fr/cazy/CAZY/index.html). 23,24 Glycoside hydrolases can also be grouped into two major mechanistic classes.…”

Selectfluor: Mechanistic Insight and Applications

“…This would be consistent with the differences observed for the glycosidases as a function of their inverting or retaining nature. [107] Since a double displacement mechanism could rationally be invoked for the retaining a-1,3-GalT, experiments were conducted to determine whether 19 was a slow inactivator of this enzyme. On the time scale of the experiments performed, no inactivation was observed, demonstrating that the enzyme does not form a covalent adduct with the fluorinated substrate 19.…”