Sedoheptulose 1,7-diphosphate, fructose

The enzyme was purified from Candida utilis in 1965 by Rosen et al. (8Q). Dried yeast was allowed to autolyze in phosphate buffer at pH 7.5 for 48 hr, and the enzyme was isolated in crystalline form from these autolysates by a procedure which included heating to 55° at pH 5.0, fractionation with ammonium sulfate, and purification on phospho-cellulose columns from which the enzyme was specifically eluted with malonate buffer containing 2.0 mM FDP. Crystallization was carried out by addition of ammonium sulfate in the presence of mM magnesium chloride. The Candida enzyme was more active than the mammalian FDPases at room temperature and pH 9.5 the crystalline protein catalyzed the hydrolysis of 83 /nnoles of FDP per minute per milligram of protein. The enzyme was completely inactive with other phosphate esters, including sedoheptulose diphosphate, ribulose diphosphate, and fructose 1- or fructose 6-phosphates. Nor was the activity of the enzyme inhibited by any of these compounds. Optimum activity was observed at concentrations of FDP between 0.05 and 0.5 mM higher concentrations of FDP (5 mM) were inhibitory. 

Sedoheptulose Diphosphate. The transaldolase reaction requires phos-phoglyceraldehyde, which has become available as a sjmthetic compound only recently. A convenient method for supplying those phosphate is to add fructose diphosphate and aldolase. When this device was used in a study of the transaldolase reaction, the reaction products of the system containing both aldolase and transaldolase, and hexose diphosphate and sedoheptulose phosphate were expected to include tetrose phosphate, as shown in equation (VI) above. Tetrose phosphate failed to accumulate, however. Instead, sedoheptulose-1,7-diphosphate was found to accumulate as a result of condensation of the tetrose ester with dihydroxy-acetone phosphate in the presence of aldolase. This diphosphate reacts rapidly with aldolase, and it is not known whether it can react in any other systems, or only shuttles back and forth in response to changes in tetrose phosphate level. 

Sedoheptulose diphosphate is formed from sedoheptulose 7-phosphate and D-fructose 1,6-diphosphate in the presence of the enzymes aldolase and transaldolase (228). One of its two phosphate groups is relatively labile... 

Sedoheptulose 7-phosphate was found to be converted to dehydroshikimic acid about as well as various phosphorylated hexoses. When the heptose phosphate was incubated together with fructose 1,6-diphosphate the degree of conversion was doubled. This led to the suggestion that a reaction product, sedoheptulose 1,7-diphosphate might be the more direct substrate. Test of sedoheptulose 1,7-diphosphate yielded a very high conversion to dehydroshiki c acid (20%). When DPN was added to this, the degree of conversion was almost quantitative (80 %). Since the synthesis of dehydroshikimic acid from sedoheptulose diphosphate involves the removal of two hydrogens as well as two molecules of water, participation of a pyridine nucleotide is understandable. 

At first two molecules of triose phosphate combine to form hexose diphosphate fructose diphosphate), which is then hydrolyzed by a specific phosphatase to fructose 6-phosphate and phosphate. Hexose phosphate and triose phosphate interact in a transketolase reaction to produce erythrose 4-phosphate and xylulose 5-phosphate, which then rearranges to ribulose 5-phosphate (the first pentose molecule). In a type of aldol condensation, erythrose phosphate and triose phosphate combine to form sedoheptulose diphosphate (C4 - - C3 = C7), which is subsequently dephos-phorylated. The enzyme transketolase then transfers a C2 fragment from heptulose to triose phosphate yielding 2 moles of pentose xylulose 5-phosphate and ribose 5-phosphate) both must be rearranged to ribulose 5-phosphate. Having ribulose 5-phosphate available, the cycle can commence again first, phosphorylation with ATP to the diphosphate, then acceptance of CO2 by the diphosphate, and production of 2 moles of phosphoglyceric acid. 

The following abbreviations have been employed FDNB, 2,4-fluorodinitro-benzene F6P, fructose 6-phosphate FDP, fructose 1,6-diphosphate FDPase, fructose-1,6-diphosphatase NEM, iV-ethylmaleimide PFK, phosphofructokinase PLP, pyridoxal phosphate SDP, sedoheptulose 1,7-diphosphate SDS, sodium dodecyl sulfate. 

PG = 3-phosphoglyceric acid G3P = glyceraldehyde 3-phosphatc DHAP = dihydroxyacetone phosphate FDP = fructose 1,6-diphosphate F6P = fructose 6-phosphate G6P = glucose 6-phosphate E4P = erythrose 4-phosphate X5P = xylulose 5-phosphate SDP = sedoheptulose 1,7-diphosphaie S7P = sedoheptulose 7-phosphate R5P = ribose 5-phosphate Ru5P = ribulose 5-phosphate RuDP = ribulose 1,5-diphosphate... 

D-ifereo-Pentulose (u-xylulose, tentatively identified as the osazone) was found chromatographically to be among the products of the action of a pea enzyme on glycolaldehyde and triose phosphate (prepared from D-fructose 1,6-diphosphate)similarly, 6-deoxy-D-fructose and 6-deoxy-L-sorbosc resulted from DL-lactaldehyde, sedoheptulose from n-erythrose, " 5-deoxy-D-triose phosphate, and D-fdo-heptulosan from n-xylose. ... 

Abbreviations used Gal-l-P, galactose-l-phosphate UDPG, uridine diphosphate glucose G-l-P, glucose-l-phosphate G-6-P, glucose-6-phosphate F-6-P, fructose-6-phosphate F-l,6-P, fructose-1,6-diphosphate G-3-P, glyceraldehyde-3-phosphate DHAP, dihydroxyacetone phosphate F-l-P, fructose-1-phosphate PG, 6-phospho-gluconic acid R-5-P, ribulose-5-phosphate S-7-P, sedoheptulose-7-phosphate. 

Fig. 4. Radioautogram of products of 60 seconds photosynthesis with C 0-. Radioautograph of two-dimensional paper chromatogram of products formed by Chlorella pyrenoidosa during 60 seconds of photosynthesis with C 0 . Abbreviations P, POaH" UDPG, uridine diphosphoglucose PGA, 3-phosphoglyceric acid PEPA, phosphoenolpyruvic acid. Sugar diphosphate includes ribulose-l,5-diphosphate, sedoheptulose-1,7-diphosphate, and fructose-1,6-diphosphate.

Rabbit muscle aldolase can attack three different substrates, fructose-1,6-diphosphate, sedoheptulose-1,7-diphosphate, and fructose-1-phosphate. Although the cleavage rates of the first two substrates are almost equal, that of fructose-1-phosphate is slow. The phosphate in position 1 is thought to be indispensable for activity, and that in position 6—although dispensable—activates the enzyme activity. 

An alternative reaction to the hydrolysis of fructose 1,6-diphosphate is the condensation of glyceraldehyde 3-phosphate and erythrose 4-phosphate to form sedoheptulose 1,7-diphosphate, which is hydrolyzed by a specific phosphatase. However, this would not account for the formation of stan and other assimilation products. 

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