Fructose 1, 6-diphosphate hydrolysis

It is now generally recognized that an important site of regulation of both glycolysis and gluconeogenesis is at the level of fructose diphosphate formation and hydrolysis (10). In the direction of glycolysis, the activity of phosphofructokinase is inhibited by ATP and citrate, and this inhibition is reversed by AMP (11). The discovery that FDPase... 

P 11.91%, O 55.35%. Present in animal tissues as an equilibrium mixture with glucose-6-phosphate. The glucose-6-phosphate may be reversibly transformed into Iructose-6-phosphate by the enzyme phosphohexose isomerase. Prepn by hydrolysis of 1,6-fructose diphosphate with dil acid Neuberg, Biochem. Z. 88, 432 (1917) Ger, pat. 334,280 (Bayer) Chem. Zenfr. 1921, II, 961 Frdl. 13, 948. Role jn metabolic regulation and heat generation E. A. News-... 

This ester was first prepared 209) by partial hydrolysis of D-fructose diphosphate and later isolated by Robison 210) from the hexose monophosphate fraction obtained by yeast juice fermentation. 

Extracts of pea leaves catalyze a rapid hydrolysis of fructose diphosphate to fructose-6-phosphate (131). The enzyme has been purified from spinach by Racker (297). 

The presence in mammalian liver of a specific phosphatase which catalyzes the hydrolysis of fructose 1,6-diphosphate (1) was first reported by Gomori in 1943 ( ). He succeeded in separating the enzyme from other phosphatases present in mammalian tissues and thus clarified much of the confusion which had previously existed regarding the specificity of these phosphatases. The specific fructosediphosphatase (FDPase) was shown to require a divalent cation such as Mg2+ and to be inactive at acid or neutral pH. It was present in the livers and kidneys of a number of mammalian species. 

The hydrolysis of fructose 1,6-diphosphate occurs at the oxygen-phosphorus linkage of the substrate, leading to the formation of l80-labeled inorganic phosphate when the hydrolysis is carried out in Ha180. No evidence for an enzyme-phosphate intermediate could be obtained 22). 

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. 

Hydrolysis of ATP )delds adenosine diphosphate (ADP), an inorganic phosphate group (Pj), and energy (Figure 21.3). The energy released by this hydrolysis of ATP is then used to drive biological processes, for instance, the phosphorylation of glucose or fructose. 

We have developed preparative enzymatic syntheses of several unusual hexoketoses using fructose-1,6-diphosphate aldolase (FDP-aldolase, E.C.4.1.2.13) as catalyst and dihydroxyacetone phosphate (DHAP) and an aldehyde as substrates (15). The enzyme appears to be very specific for DHAP but will accept a variety of aldehydes as acceptors. The ketose-1-phosphates prepared are converted to the phosphate free ketoses after removal of the phosphate group by acid- or phosphatase-catalyzed hydrolysis. The ketoses can be isomerized stereospecifically to aldoses catalyzed by glucose isomerase (E.C.5.3.1.5.) from Flavobacteriuum arborescens. The equilibrium mixtures of aldoses and ketoses are then separated by chromatography on Dowex 50 (Ba ) or Dowex 1 (HSO "). Figure 1 illustrates the preparation of a mixture of 6-deoxy-6-fluoro-D-fructose... 

The phosphate group at carbon 1 of D-fructose 1,6-diphosphate is hydrolyzed in 1 AT acid, at 100°C., approximately 12 times as fast as that at carbon 6. Although at this temperature some destruction of the D-fructose occurs, a fairly good yield of the ester can be obtained by this method. An improved procedure whereby the hydrolysis is carried out at a lower temperature is given by Neuberg, Lustig, and Rothenberg 211). 

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. 

In rat liver, the phosphorylation of fructose by means of ATP occurs at a greater rate than that of glucose. It is inferred that fructokinase predominates in this tissue, and this is supported by the observation that the anaerobic degradation of glucose by rat liver slices is relatively slow. An active fructokinase has been isolated from this tissue, and in contrast to yeast hexokinase, phosphorylates at Ci. The product, fructose-1-phosphate, had previously been isolated after autolysis of liver, during fructose utilization in liver or intestine, or after enzymatic hydrolysis of fructose 1,6-diphosphate by bone phosphatase. It may be noted that both fructose-l-phosphate and fructose-6-phosphate are formed in the latter procedure. The phosphate linkage at Ci is far more labile to acid than is that at Ce. 

Hexose Diphosphate.— Fructofuranose-1 6-diphosphoric acid, (Harden-Young ester), CsHjo04(HgP04)2, is formed in the first stage of the fermentation of glucose, mannose or fructose by yeast. The phosphate on hydrolysis by boiling yields fructose, and its formation is an example of the interconvertibility of the three monosaccharides. It is also an early product in the conversion of glycogen into lactic acid, in muscle. 

Fructose Monophosphate.— This ester has been obtained by Neuberg from the 1 6-diphosphate by partial hydrolysis. Unlike the diphosphate, it is fermented by yeast. Neuberg s ester is one of the two hexose phosphates found in muscle. 

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