Inverting glycosidase

B Hydrolysis mechanism of inverting glycosidase (R = saccharide or alcohol) - single inversion 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 5.1 Minimal kinetic scheme for an inverting glycosidase k+i is the bimolec-ular rate constant for enzyme-substrate (ES) combination, k i is the unimolecular rate constant for loss of the substrate from the enzyme and k+2 is the first-order rate constant for the chemical step in the ES (Michaelis) complex. Product loss is assumed to be fast and isomerisations of the Michaelis complex to be either non-existent or rapid and reversible.

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). 

Table 5.1 Inverting ( -glycosidases, their catalytic groups and stereochemistry of protonation. The substrate stereochemistry is given as ring size p or /), with p further amplified by whether the leaving group is axial (a) or equatorial (e).

These mutants are effectively converted into inverting glycosidases which use opposite anomeric-configured donor substrates compared to their wild type glycosidases. 

In this manner, both /3- and a-D-xylosyl fluoride generate a-D-xylose, in the former instance by stereochemical inversion and in the latter by stereochemical retention. The series of events is reflected in each of the inverting glycosidases listed above. The reactions are unique not only because they involve hydrolysis of both (albeit truncated) substrate anomers, but the enzymes also catalyze an uncommon nonhydrolytic condensation reaction using the activated glycosyl fluoride. 

The inverting glycosidases operate slightly differently from the retaining glycosidases. The key catalytic residues are usually a pair of carboxylic acids, but they are typically separated by a distance of 6-12 A,... 

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