β-Glucosidases: Mechanism and Inhibition

The generally accepted mechanism of action of glycosidases which hydrolyse glycosides with overall retention of configuration at the anomeric center involves a double displacement. Initial general acid-catalyzed generation of a glycosyl-enzyme intermediate is followed by its general base-catalyzed hydrolysis. Both the formation and the hydrolysis of the glycosyl-enzyme can be considered to proceed via oxocarbonium ion-like transition states. Destabilization of such transition states can be achieved by replacing the C-2 hydroxyl of the substrate by the more electronegative fluorine, thus slowing both steps. Simultaneous incorporation of an excellent leaving group (fluoride or dinitrophenolate) as the aglycone permits the accumulation of the intermediate which is sufficiently stable to be isolated. Investigation of such an intermediate generated on a β-glucosidase, by means of 19 F-NMR, allowed its identification as an α-D-glucopyranosyl-enzyme. Activated 2-deoxy-2-fluoroglycosides therefore act as mechanism-based inactivators, thereby representing a new class of "suicide" inactivators for glycosidases.