Sucrose-6-phosphate synthetase

12 Sucrose Phosphate Synthetase (Salvuccl and Cratts-Brandner, 1991) 

Sucrose phosphate synthetase catalyzes the reaction of UDP-glucose with fructose-6-P to form sucrose-6-P and UDP. This step is the penultimate step in the synthesis of sucrose in leaves. The chromatographic method can be applied to many UDP-glucose-requiring enzymes. The method eliminates the need for treatment of reaction mixtures with alkaline phosphatase. 

Radiolabeled UDP-glucose and sucrose-P were separated on a Selectispher-10 boronate column (5 mm x 250 mm). The mobile phase was 0.12 M sodium phosphate (pH 7.6) delivered at a rate of 1 mL/min. The column eluent was monitored for absorbance at 262 nm, and for radioactivity by a radioactive flow-through detector. 

Sources of enzyme were tobacco leaf tissue or red beet tubers homogenized at 4°C in 50 mM Hepes-KOH (pH 7.2), 5 mM MgCl2,1 mM EDTA, 25 mM mercaptoethanol, 1% (w/v)polyvinylpyrollidone-40,1 mM phenylmethylsulfo-nyl fluoride, and 10 fiM leupeptin. Supemates obtained by centrifugation were desalted and equilibrated with a buffer containing all but the last three components in the homogenization medium. 

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Salerno, G.L., Gamundi, S.S., and Pontis, H.G., A procedure for the assay of sucrose synthetase and sucrose phosphate synthetase in plant homogenates, Anal. Biochem., 93, 196-199, 1979. 

Although the bundle sheath chloroplasts contain all the enzymes of the RPP cycle, there is now evidence that some of the 3-PGA formed by the activity of rubisco is exported to the mesophyll cells [9]. Bundle sheath chloroplasts of maize are deficient in photosystem II activity and apparently cannot produce sufficient NADPH to reduce all of the 3-PGA formed to triose phosphate. Responsibility for this step is thus shared with the mesophyll chloroplasts which recycle triose phosphate to the bundle sheath as DHAP. This transport of 3-PGA from bundle sheath to mesophyll permits the synthesis of sucrose in the mesophyll cell cytoplasm. The evidence suggests that the mesophyll cells are the major site of sucrose synthesis [10-13]. Sucrose phosphate synthetase, one of the regulatory enzymes of sucrose synthesis and fructose 6-phosphate, 2-kinase (Fru-6-P,2K), the enzyme synthesizing fructose 2,6-bisphosphate — a potent regulator of cytoplasmic sucrose synthesis (see Section 5.4.1) — are both almost completely confined to the mesophyll cells. 

Sucrose can be synthesized from (a) UDPG and fructose and (b) UDPG and fructose-6-phosphate. In the latter case the product is sucrose phosphate which can be cleaved by a phosphatase to sucrose and phosphate. The enzymes catalyzing the synthesis are called sucrose synthetase (1) and sucrose phosphate synthetase (2), depending on the product. 

Sucrose-phosphate synthetase resides exclusively within the chloro-plast and may be concerned with the initial synthesis of sucrose whereas sucrose synthetase is more abundant in non-photosynthetic tissues and may be primarily involved in the metabolism of translocated sucrose rather than its direct synthesis in association with photosynthetic reactions. 

Results obtained by Leloir and Cardini indicated that two separate enzymes are involved in the biosynthesis, in plants, of sucrose and sucrose phosphate from D-fructose and D-fructose 6-phosphate, respectively in the presence of UDP-D-glucose these enzymes have been partially separated. Slabnik and coworkers succeeded in isolating sucrose synthetase and sucrose 6-phosphate synthetase from potato tubers, and determined some of the properties of the partially purified preparations. The sucrose synthetase showed an optimum activity at 45 and was inhibited completely by ADP and some phenolic D-glucosides, whereas these had no effect on sucrose 6-phosphate synthetase. 

Five of the enzymes of UMP biosynthesis exist in the soluble fraction of Ehrlich ascites carcinoma as two enzyme complexes [143]. One complex contains the first three enzymes of the pathway, carbamoyl phosphate synthetase, aspartate carbamoyltransferase and dihydro-orotase and has an apparent molecular weight of 800000 to 850000 daltons. The second enzyme complex contains orotate phosphoribosyltransferase and orotidylic acid decarboxylase and sediments in a sucrose gradient with 30% dimethyl sulphoxide and 5% glycerol with an apparent molecular weight of 105 000 to 115000 daltons [143]. 

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