Inverting glycosidases, enzyme

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.

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). 

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. 

Examples of the retaining enzymes are p-galactosidase, invertase and lysozyme. Inverting glycosidases, such as trehalase and p-amylase, have been used for the synthesis of alkyl glycosides to a lesser extent. 

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. 

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