A unique demonstration is presented of the capacity of glycosidases to create anomeric configuration de novo. Purified Candida tropicalis α-glucosidase and sweet almond β-glucosidase have been found to attack the same substrate, d-glucal, and to convert this unusual glycosyl substrate (which lacks α or β anomeric configuration) to 2-deoxy-α- (or β-) d-glucose, respectively. The stereospecificity of the hydration reaction catalyzed by each enzyme in D2O was revealed by the use of high-resolution (270 MHz) 1H magnetic resonance spectroscopy. The α-glucosidase caused a specific axial protonation (deuteration) of d-glucal at C-2, and formation of 2-deoxy-α-d-[2(a)-2H]glucose. The β-glucosidase catalyzed an oppositely directed axial protonation at C-2 and formation of 2-deoxy-β-d-[2(e)-2H]glucose. These results are not accounted for by the generally accepted mechanisms of carbohydrase action derived from studies with glycosidically linked substrates alone. d-Glucal apparently binds to the enzymes with essentially the same overall orientation as the d-glucosyl moiety of glycosidically linked substrates (with the double bond of d-glucal lying essentially in the plane of the similarly bound d-glucosyl group). Thus, the α-glucosidase evidently protonates d-glucal from above the double bond and α-d-glucosidic substrates from below the glycosidic oxygen; β-glucosidase apparently protonates d-glucal from below the double bond and β-d-glucosides from above the glycosidic oxygen. A detailed mechanism is proposed for the hydration of d-glucal by each enzyme, involving an incipient glycosyl carbonium ion and assuming the presence at the active site of two carboxyl groups arranged to account for catalysis of glycosylations from glycosidically linked substrates. That d-glucal serves as a glycosyl substrate for these enzymes strongly supports the concept that glycosidases and glycosyltransferases are catalysts of glycosylation (i.e., glycosylases), since this concept does not make the usual assumption that carbohydrases are restricted to acting on substrates having a glycosidic bond and either α- or β-anomeric configuration.
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