Retaining glycosidases hydrolysis

A Hydrolysis mechanism of retaining glycosidase (R = saccharide or alcohol) - double inversion at the anomeric centre. 

Scheme 5.3 Hydrolysis and transglycosylation of a retaining glycosidase. LG = leaving group, to emphasise that activated donors such as nitrophenyl glycosides or fluorides are often used.

Figure 5.38 Burst kinetics showing liberation of aglycone during the establishment of a steady state during hydrolysis of a snbstrate by a retaining glycosidase, when the hydrolysis of the glycosyl-enzyme is rate determining. The glycone was fluorinated, so the experiment was performed in a conventional spectrometer (note time-scale). Note the burst of 0.03 absorbance units.

As might be expected, in the absence of acceptors the 2 -deoxyribosyl-enzyme slowly hydrolyses intriguingly, however, the hydrolysis is associated with the production of substantial quantities of ribal, which is then slowly hydrolysed. Formation of ribal is clearly the near microscopic reverse of the first step in the hydration of glycals by retaining glycosidases (Figure 5.21), for some reason favoured with a 2-deoxy substrate. 

As discussed above (Section 2.2.2) the hydrolysis reactions of glycosides and methoxymethyl acetals involve nucleophilic participation by water. If water can act as a nucleophile in these systems then reactions with other, better nucleophiles are to be expected. (The model, and the inspiration for most work in this area is the enzyme lysozyme which - like retaining glycosidases in general - uses two carboxyl groups, one acting as a nucleophile, the other as a general acid, to accomplish (exo-... 

Figure 1. Retaining glycosidase mechanism showing the transition states of the glycosylation and deglycosylation steps (here illustrated for the hydrolysis of aryl P- lactosides by Tr Cell A).

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