Glycosidase, inverting, mechanism

Fig. 7. Retaining and inverting mechanisms of glycosidases Only one of the dashed bonds (jr...

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. 

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

Figure 19 Mechanism of /3-glycosidase active site labeling with probes for (a) retaining and (b) inverting /3-glycosidases.

Great care must be exercised before attributing molecular significance to values of and K. The minimal kinetic mechanism for an inverting glycosidase is that of Scheme 5.1, which leads to the expressions for and kcs,tlKi-n in eqns (5.2), (5.3) and (5.4). 

Slow proton transfer makes possible the occurrence of iso mechanisms -mechanisms in which the form of the enzyme released after catalysis is different to that at the start of the cycle. A candidate would be any inverting glycosidase, which is released with the acid group deprotonated and the basic group protonated [Figure 1(b)], although no example in the glycosyl transfer area has yet been demonstrated (the best example is proline racemase, " in which two cysteines act, one as an acid and the other as a base). 

Scheme 3 General glycosidase mechanisms, (a) An inverting /3-glycosidase. (b) A retaining /3-glycosidase proceeding through an intermediate with a 4C1 conformation, (c) Substrate-assisted mechanism.

Scheme 3 General mechanism utilized by inverting glycosidases.

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