Ribulose- 1,5-diphosphate

Binding of C02 takes place in aqueous medium by the carboxylation reaction of ribulose-diphosphate (RuDP) with the formation of 3-phospho-glycerine acid (PGA) - table 5. Water molecule and radical C=0 at the distances of molecular interaction have quite similar values of PE-parameters for forming the general structural grouping of dimeric composite type. Total PE-parameter of water molecule and radical C=0 hearly equals PE-parameter of C02 and therefore the molecules of C02 and H20 join RuBP with the formation of two radicals COOH b PGA (table 5). In ferment RuDP- carboxylase, Mg atoms and 0" ions (5.4867 eV and 4.755 eV) play an active role, their PE-parameters similar to PE-parameter of radical COOH. 

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. 

The neutral FDPase and SDPase activities, which were present in the crude spinach extracts, were precipitated at lower ammonium sulfate concentration and could thus be separated from the specific alkaline FDPase. These activities appeared to be associated with the chloroplast fraction and did not require the presence of a divalent cation for activity. In crude extracts only the alkaline FDPase activity was inhibited by antiserum prepared by immunizing rabbits with the purified alkaline FDPase. The neutral FDPase was also active with ribulose diphosphate (RuDP) (98). 

A similar alkaline FDPase has also been obtained in highly purified form from Euglena gracilis (99) by heating and fractionation on DEAE-cellulose. The specific activity of the best preparation was approximately one-half that reported by Racker and Schroeder for the spinach enzyme. The enzyme appeared to be specific for fructose diphosphate, although SDP and ribulose diphosphate were not tested. The enzyme also required Mg2+ and was most active at pH 8.3 it showed very little activity at pH 7.5 or below. 

All enzymes exhibit various features that could conceivably be elements in the regulation of their activity in cells. All have a characteristic pH optimum which makes it possible for their catalytic rates to be altered by changes in intracellular pH (e.g. in ribulose diphosphate carboxylase(l7>). The activities also depend on the concentration of substrates, which may vary according to intracellular conditions. Moreover, many require metal ions or vitamins, and the activity of enzymes may be a function of the concentrations in which such materials are present (e.g. the effect of iron limitation on the citric acid fermentation of Aspergillus niger ). However, over and above these factors, some enzymes have other properties that... 

I. Storroe, and B. R. McFadden, Ribulose diphosphate carboxylase/oxygenase in toluene-permeabilized Rhodospirillum rubrum, Biochem. J. 1983, 212, 45-54. 

Ribulose diphosphate takes, a mole of CO2 Gives two 3-phosphoglycerates, (if Calvin s story s true) NADPH now provides, reducing power to make Two phosphoglyceraldehydes (as ATP we break). 

Note that six ribulose diphosphates (RuDP) combine with six COj to yield twelve 3-phosphoglycerates (3PG). Two of these give rise to glucose the other ten eventually regenerate six RuDP to continue the fixation process. 

Let us return to the so-called dark reaction of photosynthesis. In it, both the NADPH2 and the ATP formed in the light reactions are consumed in the fixation of C02. The fixation reactions were charted by Melvin Calvin and his co-workers in Berkeley, California (for which Calvin received the Nobel Prize for 1961), with the use of radioactive carbon dioxide. During these reactions, C02 is made to combine with a pentose (5-carbon) sugar, ribulose diphosphate, to give an unstable 6-carbon intermediate which breaks down to two molecules of the 3-carbon phosphoglyceric acid. 

Ribulose diphosphate carboxylase Succinic dehydrogenase Superoxide dismutase Tyrosine aminotransferase Xanthine dehydrogenase Xanthine oxidase... 

Overall, the hydrogen stored in NADPH is used to reduce C02 to carbohydrate units (0H2O). This is not a direct reaction because the C02 is first combined with a C5 compound, ribulose diphosphate (RDP), which then spontaneously splits into two identical C3 molecules, phosphoglyceric acid (PGA). Most of the PGA is used to synthesize further RDP but some is reduced by NADPH, using energy supplied by the ATP/ADP system, to give triose phosphate, which in turn is converted into the glucose phosphate from which various carbohydrates are synthesized. This assimilatory path is known as the Calvin cycle and is involved in all autotrophic carbon fixation, whether photosynthetic or chemosynthetic. 

Some subaerial plants use a different pathway, involving carboxylation of phosphoenol pyruvic acid (PEP) instead of ribulose diphosphate (the C02 is actually transformed into bicarbonate before incorporation), which subsequently forms a C4 compound, oxaloacetic acid, instead of PGA (Fig. 1.7). Consequently such plants are termed C4 plants. The C4 path is a relatively recent evolutionary development of particular advantage in hot dry climates (see Box 1.10).The PEP cycle effectively transfers C02 to the Calvin cycle, and each cycle confers an isotopic fractionation. Some plants, the CAM plants (see Box 1.10), can use the combined PEP-Calvin cycle path (with some leakage of C02 out of the cell between the cycles) or just the Calvin cycle. The effects of these pathways on the overall isotopic fraction are reflected in the 813C values in Table 5.8. 

Carboxylase, ribulose diphosphate (Nicotiana sylvestris ctone NySS4 small subunit precursor reduced) 2236a, 3973, 4249... 

Deoxyribonucleic acid Nicotiana syivestris clone NySS4 ribulose diphosphate carboxylase small subunit gene) 950b, 1919a, 4249... 

Oxygenase, ribulose diphosphate 429b, 2236a, 2878e, 4249,4641... 

Multi-Enzyme Cofactor Requiring Processes - A more complex level of applied enzymology is reached in the use of multi-enzyme schemes to synthesize complex molecules. Examples of such syntheses include ribu-lose-1,5-diphosphate, important in the study of ribulose-diphosphate carboxylase lactosamine,from the first use of the Lelolr pathway enzjnnes in synthesis and S-adenosyl-L-methionine. ... 

Bowes, G., W. L. Ogren, and R. H. Hageman Phosphoglycolate Production Catalyzed by Ribulose Diphosphate Carboxylase. Biochem. Biophys. Res. Commun. 45, 716 (1971). 

These studies were continued by Wilson and Calvin (1955), who left the light on but suddenly lowered the tension of CO2 to 0.003%. Since PGA is a product of the carboxylation reaction, it was expected that its concentration would fall. This is exactly what happened. At the same time, the concentration of ribulose diphosphate rose rapidly and then fell. This behavior is to be expected if ribulose diphosphate is a substrate for the carboxylation reaction, since lowering the CO2 pressure will stop the reaction which uses up RuDP. Thus, the carboxylation of ribulose diphosphate to give PGA as a first step in the carbon reduction pathway was discovered [Eq. (8)]. 

The carboxylation of ribulose diphosphate by cell-free extracts of Chlorella was demonstrated by Quayle et al. (1954), who named the enzyme carboxydismutase. The enzyme has been purified from spinach leaves (Weissbach et al, 1956 Mayaudon et al., 1957). The purified enzyme mediates the addition of water and CO2 to ribulose diphosphate, with an intramolecular oxidation-reduction reaction, or dismutation, leading to the formation of two molecules of PGA. In the carboxylation part of the reaction the carbon atom of CO2 becomes bonded to the number 2 carbon atom of ribulose diphosphate. 

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