Glycosidases active site carboxylates

Fig. 2.—Possible Formation of an Enzyme-linked Acylal (—X = Carboxylate) from an Aldono-1,5-lactone at the Active Site of a / -Glycosidase (after Sinnot and Souchard ).

Figure 5.13 Protonation states of a glycosidase with two active site carboxylates.

Another interesting approach is the use of glycosynthases, that are classified as mutated retaining glycosidases where the active-site carboxylate nucleophile has been replaced by a non-nucleophilic amino acid side chain (such as serine or alanine). Glycosynthases have proven to be very valuable biocatalysts for the synthesis of oligosaccharides and some excellent reviews appeared in the last years [3, 4, 213, 253-264],... 

Retaining and inverting O-glycosidases make use of a general acid/general base pair comprised of a carboxylic acid and carboxylate of aspartic acid or glutamic acid side chains (see also Eig. 21). These side chains have similar values in solution but the micro-environments of enzyme active sites allow each pK to be tuned so that each side chain is in the correct protonation state. This makes it possible for a carboxylate and a carboxylic acid to exist in the active site simultaneously. 

Recent crystal structure studies confirm the presence of two fixed and opposed active-site carboxyl groups in nearly all glycosidases, including /3-galactosidase 77 cellulases 4 of families 6,7,9,10, and 45 56 80-88 xylanases 89-93 alpha-amylases 94-100 beta-amylase 101-102 and glucoamylase.1031048 Hev-amine, a chitinase from Hevea braziliensis. is exceptional in lacking a cat-... 

Deviations from the Canon Unorthodox Catalytic Residues in Retaining Glycosidases. While the presence of two catalytic carboxylates in retaining gly-cosidase active sites is nearly universal, several notable exceptions exist. The following section presents a brief overview of these mechanistic deviants, while a number of excellent reviews are recommended as starting points for more detailed discussions (56,57,66,68). 

---