The Biosynthesis of Sucrose - Correction

Leloir, Luis F.; Cardini, Carlos E.

doi:10.1021/ja01120a666

Cited by 25 publications

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“…The biosynthesis of sucrose-P was first reported by Leloir and Cardini (14) to occur via transfer of glucose from UDPglucose to fructose-6-P. Hawker and Hatch (10) and Hawker (7) subsequently reported the presence in plant tissues of a specific sucrose-6-P phosphatase. Earlier labeling studies (1,3,4) suggested that, in leaves, sucrose was synthesized from hexose phosphates and not from free sugars in the cell, and that implicated sucrose-P synthase as the enzyme responsible for sucrose synthesis in plants.…”

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Kinetic Characterization of Spinach Leaf Sucrose-Phosphate Synthase

Amir

Preiss²

1982

Plant Physiol.

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The spinach (Spinaca oeracca) leaf sucrose-phosphate synthase was partially purified via DEAE-cellulose chromatography, and its kinetic properties were studied. Fructose-6-phosphate saturatio curves were sigmoidal, while UDPglucose saturation curves were hyperbolic. At The biosynthesis of sucrose-P was first reported by Leloir and Cardini (14) to occur via transfer of glucose from UDPglucose to fructose-6-P. Hawker and Hatch (10) and Hawker (7) subsequently reported the presence in plant tissues of a specific sucrose-6-P phosphatase. Earlier labeling studies (1, 3, 4) suggested that, in leaves, sucrose was synthesized from hexose phosphates and not from free sugars in the cell, and that implicated sucrose-P synthase as the enzyme responsible for sucrose synthesis in plants.The enzyme isolated from wheat germ has been highly purified and studied in great detail (18,23). Some of its kinetic properties have been postulated to be pertinent to its regulation in vivo (19, 23). However, very little has been published on the kinetic properties of the leaf sucrose-P synthase, even though the enzyme activity has been detected in various leaves (2,6,9,13,16,17,20).The following describes a partial purification of the spinach leaf sucrose-P synthase and its preliminary kinetic characterization. Emphasis of the studies were directed to determining possible regulatory modes of the enzymic activity. Hepes (pH 7.5); 20 mmi f-mercaptoethanol; 2 mm EDTA and 2% ethyleneglycol. The extraction was carried out in a vacuum flask under an atmosphere of N2. After the extract had been squeezed through a cheese cloth, it was centrifuged at 16,000g for 20 min, and the supernatant fluid was saved. This solution was poured onto a DEAE-cellulose column (2.5 x 20 cm), previously equilibrated with a buffer solution, containing 20 mm Hepes-NaOH (pH 7.5), 10%o ethyleneglycol, 2 mm Na2EDTA, and 5 mM f,-mercaptoethanol. The column was washed with about 200 ml of the above buffer, and the enzyme was eluted with a 500 ml linear NaCl gradient (0 to 0.5 M) in the same buffer. The fractions with the sucrose-P synthase activity were pooled and concentrated in an Amicon TCF 10 thin-channel cell with a PM-10 membrane. After concentration to about 5 ml, the enzyme fraction was diluted to 20 ml with the above DEAE cellulose equilibration buffer. The enzyme was frozen and stored in liquid N2.Enzyme Assays.Assay of Sucrose-P Synthase. The reaction mixture contained the following: 5 ,umol Hepes-NaOH (pH 7.5); 1.2 ,Amol fructose-6-P; 1.2 umol UDP-["4C]glucose (specific activity, 100,000 to 400,000 cpm/4mol); 0.2 mg BSA; 0.5 ,umol MgCl2; and enzyme; in a total volume of 0.125 ml. After 20 min at 37°C, the reaction was stopped by immersing the tubes in boiling water for 1 min. The sucrose-6-P formed was hydrolyzed to sucrose with E. coli alkaline phosphatase (0.1 unit/assay). After incubation for 30 min at 37°C, 0.4 ml of a 5% sucrose solution was added. The diluted reaction mixture was loaded onto a l-ml DEAE cellulose column and eluted with 5 ml H20...

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Kinetic Characterization of Spinach Leaf Sucrose-Phosphate Synthase

Amir

Preiss²

1982

Plant Physiol.

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“…Sucrose-phosphatase (SPP; EC 3.1.3.24) catalyzes the final step in the pathway of sucrose biosynthesis, in which sucrose-6 Fphosphate (Suc6P) is dephosphorylated to sucrose (Leloir and Cardini, 1955;Hawker and Hatch, 1966). Suc6P is produced by sucrose-phosphate synthase (SPS; EC 2.4.1.14), and the irreversible hydrolysis of Suc6P by SPP is essential to pull the reversible SPS reaction in the direction of sucrose synthesis (Lunn and ap Rees, 1990a).…”

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The Structure of a Cyanobacterial Sucrose-Phosphatase Reveals the Sugar Tongs That Release Free Sucrose in the Cell

et al. 2005

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Sucrose-phosphatase (SPP) catalyzes the final step in the pathway of sucrose biosynthesis in both plants and cyanobacteria, and the SPPs from these two groups of organisms are closely related. We have crystallized the enzyme from the cyanobacterium Synechocystis sp PCC 6803 and determined its crystal structure alone and in complex with various ligands. The protein consists of a core domain containing the catalytic site and a smaller cap domain that contains a glucose binding site. Two flexible hinge loops link the two domains, forming a structure that resembles a pair of sugar tongs. The glucose binding site plays a major role in determining the enzyme's remarkable substrate specificity and is also important for its inhibition by sucrose and glucose. It is proposed that the catalytic reaction is initiated by nucleophilic attack on the substrate by Asp9 and involves formation of a covalent phospho-Asp9-enzyme intermediate. From modeling based on the SPP structure, we predict that the noncatalytic SPP-like domain of the Synechocystis sucrose-phosphate synthase could bind sucrose-6 F -phosphate and propose that this domain might be involved in metabolite channeling between the last two enzymes in the pathway of sucrose synthesis.

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“…No sucrose synthesis occurred when fructose or fructose-1,6-diphosphate and UDPG were added to sugar beet leaf homogenates. Burma and Mortimer stated that their experiments with sugar beet leaves eliminated Leloir and Cardini's (11) first enzyme mechanism demonstrated with wheat germ, which was the interaction of UDPG and fructose for the direct formation of sucrose plus pyrophosphate.…”

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