Inverting glycosidases

Evidence for a glycosyl-enzyme intermediate of finite lifetime with inverting a-D-glycosidases, and details of its reaction, came from studies with 2,6-anhydro-l-deoxyhept-l-enitols and glycosyl fluorides. - Analysis of hydration and hydrolysis products on the one hand, and of glycosyla-tion products on the other, indicated an intermediate that could be approached by water from the yff-face only of the ring, and by other glycosyl acceptors only from the a-face (see Schemes 4 and 5 This can be considered a proof of the principle of microscopic reversibility of chemical reactions. 

B Hydrolysis mechanism of inverting glycosidase (R = saccharide or alcohol) - single inversion at the anomeric centre... 

Scheme 2 Hydrolysis mechanisms of retaining (A) and inverting (B) glycosidase and P-A-acetylhexosaminidase, which uses a modified retaining mechanism (C).

The glycosidases act by two different mechanisms which is revealed by the stereochemistry at the anomeiic centre of the product (McCarter and Withers, 1994). In one type of glycosidases the anomeiic centre is directly attacked by a hydroxide to give a product with inverted stereochemistry at the anomeiic centre. In the other mechanism, the anomeric centre is attacked by the carboxylate group of a glutamic acid residue to form an intermediate in which the carbohydrate moiety is covalently bound to the enzyme similar to in epoxide hydrolases (Figure 2.16) and serine hydrolases. Attack on this intermediate by a nucleophile leads to the net result which is retention of the stereochemistry at the anomeric centre. 

With inverting glycosidases, the anomeric configuration is changed from a in the substrate to (3 in the product, or vice versa during the hydrolysis reaction (Scheme 9.20). 

SCHEME 16.2 Glycosidase mechanisms for (a) a retaining a-glycosidase and (b) an inverting a-glycosidase proceeding through an oxocarbenium ion-like intermediate. 

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