Sucrose phosphorylase specificity

The enzyme has been found in Leuconostoc mesenteroides66 and Pseudomonas saccharophila.6 In a series of outstanding researches Doudoroff and Hassid with associates investigated the specificity requirements of sucrose phosphorylase from P. saccharophila. It was found that the enzyme exhibits an absolute specificity for the D-glucose moiety of its... 

Fig. 2 Main reactions catalyzed by sucrose-utilizing transglucosidases. The synthesis of a-glucans (Glc) linked through either a-1,6 a-1,3, a-1,4, or a-1,2 osidic linkages depending on the enzyme specificity is obtained by glucansucrases. The formation of a-D-glucose-1-phosphate from orthophosphate is obtained using sucrose-phosphorylases. The mechanism involves a double-inversion at the anomeric center via the formation of a covalent P-D-glucosyl enzyme intermediate...

Sucrose phosphorylase catalyzes reversible glucosyl transfer from sucrose to orthophosphate, although other acceptors, including As04 ", water, fructose, and a number of other ketoses, are equally suitable. Donor specificity is more strict and, aside from the substrate/product pair of sucrose and a-D-glucosyl phosphate, only a-D-glucosyl fluoride has been well characterized (12, 148-151). 

Bacillus subtilis levansucrase (sucrose 2,6-/8-D-fructan 6-)8-D-fructosyltrans-ferase, EC 2.4.1.10) catalyzes fructosyl transfer from sucrose to levan (154). In the absence of a fructosyl acceptor, the primary reaction is sucrose hydrolysis, although a limited amount of self-initiated levan synthesis occurs as well (155). As with sucrose phosphorylase, acceptor specificity is broad a number of saccharides and other nucleophiles are suitable fructosyl acceptors (154-158). The complete amino acid sequence of the approximately SO-kDa enzyme has been determined by both protein (159) and gene (160) sequence analyses. The three-dimensional structure at 3.8 A reveals a rod- or ellipsoid-shaped protein with a length some four times the diameter (161). 

Sucrose phosphorylase exhibits a very high degree of specificity for the glucose portion of its substrates, but it reacts mth a number of glycosyl acceptors in addition to phosphate and fructose. The enzyme from P. saccharophUa, but not from P. putrefadens, will react with sorbose. 

This enzyme is very specific it does not react with sorbose, fructose phosphates, glucose, or any other compound tested in place of fructose, and glucose-l-phosphate cannot replace UDPG. Unlike the case of sucrose phosphorylase, the equilibrium of this reversible reaction favors sucrose synthesis, and the presence of this enzyme in the cells of higher plants implicates this reaction in sucrose synthesis. 

The fact that sucrose can be synthesized from -D-glucose 1-phosphate and D-fructose is consistent with the evidence presented by Isbell and Pigman 172) and Gottschalk 173) that D-fructose exists as an equilibrium mixture of the pyranose and furanose forms. The total system of sucrose synthesis can be represented by the equation (Fig. 4) which includes the equilibrium reaction of the two ring forms of D-fructose. The occurrence of D-fructose in the sucrose molecule as D-fructofuranose is an indication that sucrose phosphorylase is specific for the furanose configuration of that ketose 17A, 175),... 

Extracts of Neisseria perflava contain phosphorylase also, but this could be differentiated from amylosucrase, which is specific for sucrose and has no action on a-D-glucosyl phosphate. The polysaccharide has been investigated by methylation and enzymic analysis, and the presence of branched chains of (1— 4)-linked a-D-glucose residues rigidly established. The acceptor specificity of amylosucrase has not been studied in detail, but maltosaccharides and small amylose-type molecules are logical acceptors, especially as amylosucrase action on sucrose is inhibited by a-amylase. 

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