Scope and Mechanism of Carbohydrase Action. Stereospecific Hydration of 2, 6-Anhydro-L-Deoxy-D-Gluco-Hept-L-Enitol Catalyzed by α- and β-Glucosidases and an Inverting Exo-α-Glucanase

D-Glucosylases of several types have been found to catalyze specific hydration and glycosyl transfer reactions (the former predominating) when incubated with 2, 6-anhydro-1-deoxy-D-gluco-hept-1-enitol. α-Glucosidases from Candida tropicalis and from rice convert this substrate into 1-deoxy-α-D-gluco-heptulose. The β-glucosidase from sweet almonds and an exo-1, 6-glucanase (glucodextranase) from Arthrobacter globiformis convert the substrate to 1-deoxy-β-D-gluco-heptulose. The anomeric form of the sugar was identified by following ¹H NMR spectra of enzyme-enitol digests. The C-1 methyl group protons of the α and β anomers resonated at 1.45 and 1.40 ppm, respectively. Nonenzymatic anomerization of the β to the α form of the sugar was noted and, at equilibrium, ¹³C and hydroxyl ¹HNMR spectra showed the presence of only the α anomer. The present work provides the first demonstration of the ability of an “inverting” glucanase to create specific anomeric configuration de novo from a substrate lacking α or β configuration. Indeed, the actions of glucodextranase on this enolic glycosyl donor lead not only to 1-deoxy-β-D-gluco-heptulose but also to a 1-deoxy-α-D-gluco-heptulosyl transfer product when enitol is the acceptor. These results reveal a catalytic mechanism that is able to convert a substrate to products of either configuration, depending on the type of acceptor. A mechanism is proposed for the stereospecific hydration of 2, 6-anhydro-1-deoxy-Dgl uco-hept-1-enitol 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 reactions with appropriate glycosidic substrates. These observations provide strong evidence for the functional flexibility of the catalytic groups of glycosylases.