Activated sugar-nucleotide substrate

The most frequently applied concepts for the enzymatic formation of oligosaccharides and glycoproteins involve the utilization of glycosyl transferases or exo- and e do-glycosyl hydrolases (glycosidases) [58]. Glycosyl transferases require expensive and often unstable sugar nucleotides as activated donor substrates, a major problem besides the poor availability of the enzymes. However, trans-... 

In this context, the use of enzymes has emerged as a practical alternative to chemical synthesis [6-13]. Several examples have been reported that are based on the use of Leloir type-glycosyltransferases, which are mostly membrane-associated and act on nucleotide-activated sugars as donor substrates. However, the use of these enzymes in vitro still remains limited by the difficulty of enzyme purification and by their need for expensive sugar-nucleotides [6]. Re-engineering of microbial cells producing these proteins appears to be promising for the synthesis of specific carbohydrate stmctures [14]. 

There are a number of cofactor independent carbohydrate epimerases that act on activated substrates, such as keto-sugars and keto-sugar nucleotides, although there is a paucity of details about their mechanisms. D-ribulose-5-phosphate 3-epimerase catalyzes the stereoinversion of substrate about the C-3 carbon to form D-xylulose 5-phosphate (as in Fig. 7.15) [102, 103]. Solvent hydron is completely incorporated into the product at the C-3 carbon, during epimerization in the d-xylulose 5-phosphate to o-ribulose 5-phosphate direction [102], This was taken as evidence for a two-base mechanism. 

Epimerases and racemases may or may not employ enzyme cofactors (organic or inorganic) to activate the stereogenic center of the substrate. Common cofactor-stabilized intermediates include resonance-stabilized carbanions and metal-stabilized enolates. The substrate itself can be intrinsically activated if the stereogenic center is adjacent to a carbonyl or carboxylate group. A preponderance of racemases and epimerases act on activated substrates. A number of sugar and sugar nucleotide epimerases act on unactivated substrates. Double proton transfers may proceed, in principle, by either a one- or two-base mechanism. However, only two-base mechanisms have been observed for racemases. 

Various sugar nucleotides were found to be utilized in this reaction. For the yeast enzyme, compounds containing D-mannose were the best substrates, and, among D-mannose nucleotides, the activity increased in the order ADP- < GDP- < dTDP- < UDP-. 

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