Fructose-6-phosphate reduction

The long-known stimulating effect of mono- and polynitro com-pounds on the onset of fermentation in yeast maceration juice has been reinvestigated by Vandendriessche. The induction time is shortened significantly by 2,4- or 2,5-dinitrophenol, while 2,6-dinitro-phenol did not show such an effect. The influence is evident when using as substrates the fermentable hexoses and D-fructose-6-phosphate, but not hexose diphosphate. According to MarkoviCev a stimulation of the oxidation processes can be proved thereby. It is probable that these effects are related to the known phytochemical reduction of nitro compounds (see pp. 98 and 99). 

An analogous use of ATP is found in photosynthetic reduction of carbon dioxide in which ATP phos-phorylates ribulose 5-P to ribulose bisphosphate and the phosphate groups are removed later by phosphatase action on fructose bisphosphate and sedoheptulose bisphosphate (Section J,2). Phosphatases involved in synthetic pathways usually have a high substrate specificity and are to be distinguished from nonspecific phosphatases which are essentially digestive enzymes (Chapter 12). 

The reactions enclosed within the shaded box of Fig. 17-14 do not give the whole story about the coupling mechanism. A phospho group was transferred from ATP in step a and to complete the hydrolysis it must be removed in some future step. This is indicated in a general way in Fig. 17-14 by the reaction steps d, e, and/. Step/represents the action of specific phosphatases that remove phospho groups from the seven-carbon sedoheptulose bisphosphate and from fructose bisphosphate. In either case the resulting ketose monophosphate reacts with an aldose (via transketolase, step g) to regenerate ribulose 5-phosphate, the C02 acceptor. The overall reductive pentose phosphate cycle (Fig. 17-14B) is easy to understand as a reversal of the oxidative pentose phosphate pathway in which the oxidative decarboxylation system of Eq. 17-12 is... 

In the presence of excess phosphohexose isomerase and glucose-6-phos-phate dehydrogenase the rate of reduction of TPN is proportional to the rate of cleavage of fructose diphosphate. For cases when small, quantities of fructose diphosphate must be used, a second spectrophoto-metric assay, in which fructose diphosphate is regenerated, has been proposed 20). Fructose 6-phosphate is phosphorylated with ATP and phosphofructokinase, and the adenosine diphosphate (ADP) produced is measured with phosphoenolpyruvate and lactic dehydrogenase ... 

A reversible covalent modification that plants use extensively is the reduction of cystine disulfide bridges to sulf-hydryls. Many of the enzymes of photosynthetic carbohydrate synthesis are activated in this way (table 9.3). Some of the enzymes of carbohydrate breakdown are inactivated by the same mechanism. The reductant is a small protein called thioredoxin, which undergoes a complementary oxidation of cysteine residues to cystine (fig. 9.5). Thioredoxin itself is reduced by electron-transfer reactions driven by sunlight, which serves as a signal to switch carbohydrate metabolism from carbohydrate breakdown to synthesis. In one of the regulated enzymes, phosphoribulokinase, one of the freed cysteines probably forms part of the catalytic active site. In nicotinamide-adenine dinucleotide phosphate (NADP)-malate dehydrogenase and fructose-1,6-bis-... 

On the basis of these results a general pathway for the formation of HDF from sugars was assumed as exemplified for fructose-1,6-biphosphate in Figure 11 [88]. Elimination of the phosphate group at C-6 of the l-deoxyosone-6-phosphate, results in acetylformoine which was established as the key intermediate in HDF formation [88]. Reduction of acetylformoine either by a disproportionation reaction with a second molecule of acetylformoine or by further reductive agents present in foods, like vitamin C, then, after elimination of water, generates HDF. 

The PPC allows the generation of NADPH reduction equivalents required for cell anabolism, and ribose 5-phosphate molecules for the synthesis of nucleic acids. Alternatively, ribose 5-phosphate can also be generated or transformed into fructose 6-phosphate or glyceraldehyde 3-phosphate, providing metabolic flexibility to the cell, in order to balance the fluxes through these pathways. The flux through the PPC is related to the nucleic acid requirements for DNA duplication or RNA transcription, and could probably be controlled by the cell cycle (Wagner, 1997). 

Answer Ribulose 5-phosphate kinase, fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and glyceraldehyde 3-phosphate dehydrogenase would be inhibited. All have mechanisms requiring activation by reduction of a critical disulfide bond to a pair of —SH groups. Iodoacetate reacts irreversibly with free —SH groups. 

It was reported by Horecker and coworkers that one class of aldolases (called Class I to distinguish it from the Class II aldolase that is metal ion-dependent) could be inhibited by the addition of borohydride reducing agent to reaction mixtures containing both enzyme and substrate129,130. It was then shown for the fructose- 1,6-bis-phosphate aldolase that the inhibition resulted from reduction of the Schiff base formed between the dihydroxyacetone phosphate substrate and the -amino group of a lysine side chain, thereby compromising the ability of the lysine to participate in subsequent turnover. 

Glucose (Glc) is taken up and phosphorylated into glucose-6-phosphate (Glc6P), with consumption of ATP. Isomerization and phosphorylation afford fructose-l,6-bisphosphate (Frul,6P2), which is cleaved into two triose molecules D-glyceraldehyde-3-phosphate (GA3P) and dihydroxyacetone monophosphate (DHAP). These are equilibrated by triose phosphate isomerase as only GA3P is metabolized further, except approximately 5 mol% of DHAP that leaks out of the pathway via reduction to glycerol, which is excreted as a side-product. 

Excess glucose can enter the polyol pathway, where it is reduced to sorbitol (by aldose reductase and the reductant NADPH). Sorbitol dehydrogenase will oxidise sorbitol to fructose, which also produces NADH from NAD+. Hexokinase will return fructose to the glycolysis pathway by phosphorylating it to fructose-6-phosphate. However, in uncontrolled diabetics with high blood glucose, the production of sorbitol is favoured. 

Second, GSH functions, presumably nonenzymically, in the reduction of protein thiols which have become oxidized to mixed disulfides (803). In this latter function GSH in some cases converts inactive enzymes to active ones, or vice versa, and may thus serve as a means of metabolic control. Examples of this important possibility are glycogen synthetase D (EC 2.4.1.11) and fructose-1,6-diphosphatase (EC 3.1.3.11). The D form of glycogen synthetase is dependent for activity upon the presence of glucose 6-phosphate. The enzyme is inactivated by GSSG and reactivated by GSH (204). Mixed disulfide formation between thiols of the enzyme and GSSG leads to a decrease in affinity of the enzyme for its activator (205). 

Fig. 1. The reductive pentose phosphate cycle (RPP). The solid lines indicate reactions of the RPP cycle. The number of lines per arrow indicates the number of times each reaction occurs for one complete turn of the cycle in which three molecules of COj are converted to one molecule of G3P. Each reaction of the cycle occurs at least once. The double dashed lines indicate the principal reactions removing intermediate compounds of the cycle for biosynthesis. Abbreviations RuBP, ribulose 1,5-bis-phosphate PGA, 3-phosphoglycerate DPGA, 1,3-diphosphoglycerate, FBP, fructose 1,6-bisphos-phate F6P, fructose 6-phosphate SBP, sedoheptulose 1,7-bisphosphate S7P, sedoheptulose 7-phosphate Xu5P, xylulose 5-phosphate R5P, ribose 5-phosphate Ru5P, ribulose 5-phosphate TPP, thiamine pyrophosphate. From Ref. 1.

The preceding reactions yield two molecules of NADPH and one molecule of ribose 5-phosphate for each molecule of glucose 6-phosphate oxidized. However, many cells need NADPH for reductive biosyntheses much more than they need ribose 5-phosphate for incorporation into nucleotides and nucleic acids. In these cases, ribose 5-phosphate is converted into glyceraldehyde 3-phosphate and fructose 6-phosphate by transketolase and transaldolase. These enzymes create a reversible link between the pentose phosphate pathway and glycolysis by catalyzing these three successive reactions. 

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