Specificity of Sucrose Phosphorylase

That the same enzyme,28 sucrose phosphorylase, is involved in the reaction of L-arabinose is indicated by the following observation. When L-arabinose is added to a mixture containing the enzyme, D-glucose-1-phosphate and D-fructose, of which the last is present in insufficient concentration to give the maximum rate of sucrose formation, an increase 

In addition there is other evidence pointing to the fact that the same enzyme is involved in reactions with both D-fructose and L-arabinose. First, the relative rates of reaction with D-fructose and L-arabinose, respectively, remain constant after partial inactivation of the enzyme by heat. Second, the enzyme catalyzing both reactions is produced to a marked extent when sucrose is used as substrate for the growth of the organisms, but not when D-glucose or L-arabinose is used sucrose phos-phorylase is an adaptive enzyme. Third, on fractionation of the enzyme preparation with various concentrations of ammonium sulfate, the relative activities of the fractions are the same for both sugars. These observations indicate not only that the same enzyme is involved in both reactions but also that no additional enzyme is required for the formation of D-glucosyl-L-arabinose. 

Preparations were made by grinding a sufficient amount of dry bacteria with phosphate buffer at pH 6.64 to result, after various further 

After two fractionations between 0.33 and 0.6 saturated ammonium sulfate, several precipitations at 0.6 saturation were made throughout a period of two or three days, during which the preparation was stored in the refrigerator. Any denatured, insoluble proteins were removed by centrifugation, and a solution of the desired activity was prepared by dilution with buffer or distilled water. 

The chief loss in phosphorylase activity appeared to occur in the early stages of the fractionation the precipitated material remained active for several days in 0.6 saturated ammonium sulfate at 4°. The invertase was apparently inactivated by prolonged contact with 0.6 saturated ammonium sulfate, since it could not be recovered from the supernatant liquid after precipitation of the active material. 

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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... 

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. 

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...

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. 

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. 

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),... 

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