Scope and mechanism of carbohydrase action: stereospecific hydration of D-glucal catalyzed by α- and β-glucosidase

Abstract

A unique demonstration is presented of the capacity of glycosidases to create anomeric configuration de novo. Purifed Candida tropicalis alpha-glucosidase and sweet almond beta-glucosidase have been found to attack the same substrate, D-glucal, and to convert this unusual glycosyl substrate (which lacks alpha or beta anomeric configuration) to 2-deoxy-alpha-(or beta-) 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 alpha-glucosidase caused a specific axial protonation (deuteration) of D-glucal at C-2, and formation of 2-deoxy-alpha-D-[2(a)-2H]glucose. The beta-glucosidase catalyzed an oppositely directed axial protonation at C-2 and formation of 2-deoxy-beta-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 alpha-glucosidase evidently protonates D-glucal from above the double bond and alpha-D-glucosidic substrates from below the glycosidic oxygen; beta-glucosidase apparently protonates D-glucal from below the double bond and beta-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 alph- or beta-anomeric configuration.

A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

“…95) Typical reaction schemes selected from the hydration processes of glycosidases are depicted in Fig. 12, in which schemes A and B show the mechanisms expected for the hydration reaction by Hehre et al, 91) although the origin of the deuterium ion (D þ ) in the initial reaction is obscure. According to the oxocarbenium ion intermediate model, however, the hydration processes should be revised to be like the reaction schemes in Fig.…”

A Novel Metabolic Pathway for Glucose Production Mediated by α-Glucosidase-catalyzed Conversion of 1,5-Anhydrofructose

“…2D, our combined reaction eliminates the plateau during glucose production with a Ͼ40-fold enhancement of hydration as com- (16). B, 31AG-catalyzed trans-addition to GlcAL (17,18). In water (RϭH), 31AG produces ␣-2dGlc with newly formed axial proton (shown by italic boldfaced H).…”

“…1A) (16) and hydration at a double bond of D-glucal (GlcAL) by trans-addition to produce ␣-2-deoxyglucose (␣2dGlc; Fig. 1B) (17,18), although 13AGs cannot catalyze those reactions (17). These findings imply that 2-OH of the glycone portion (a glucose unit of nonreducing end that binds to subsite Ϫ1) is avidly recognized by 13AGs, whereas recognition of 31AGs is not as strong.…”

Conversion of inverting glycoside hydrolases into catalysts for synthesizing glycosides employing a glycosynthase strategy

“…Previously, they reported that b-amylase, an inverting GH, hydrolyzed a-and b-maltosyl ‰uorides to maltose and F - (13). It should be noted that b-maltosyl ‰uoride possesses the opposite anomeric conˆguration of the normal substrates that are hydrolyzed by the enzyme.…”

“…The Hehre resynthesis-hydrolysis mechanism is often di‹cult to detect because many enzymes recognize glycosyl ‰uoride not only as a donor but also as an acceptor (13)(14)(15)18,21,22), such as b-maltosyl ‰uoride for bamylase and b-glucosyl ‰uoride for glucoamylase. However, reliable experimental evidence for this mechanism was reported for trehalase using b-glucosyl ‰uoride as the donor and glucose or xylose as the acceptor (19,20).…”