Ribulose 1,5-diphosphate photosynthesis

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. 

The most important CO2 activation process is photosynthesis, in which solar photons drive a reaction that would otherwise be uphill thermodynamically the reduction of CO2 to carbohydrates coupled to water oxidation to O2. Many metalloenzymes are involved in these processes, the one that fixes CO2 is ribulose diphosphate carboxylase, in which an enolate anion of the sugar nucleophilically attacks the CO2 carbon. Cu(II), Mn(II), and are all present in the active enzyme, and one of these probably plays a role in polarizing the CO2, perhaps via an tj -OCO complex. 

D-Ribulose 5-phosphate is found together with D-ribose 5-phosphate as an oxidation product of n-gluconic acid 6-phosphate with a purified yeast enzyme (221-223). The D-ribulose 5-phosphate was identified as the o-nitrophenylhydrazone derivative, having a specific rotation in methanol of 50 5°. D-Ribulose 5-phosphate appears to be the precursor of D-ribose in yeast and rat liver. A D-ribulose diphosphate was detected in the products formed during the first few seconds of photosynthesis (224) ... 

The carboxylation of ribulose diphosphate is not the only pathway for the entry of carbon dioxide into plant metabolism. The finding of C -labeled malic acid in short-term photosynthesis suggests another pathway. This may be related to the reaction described by Bandurski and Greiner 60) where phosphoryl-enolpyruvate was carboxylated to yield oxal-acetate. Pathways of carbon dioxide fixation known to occur in other organisms 3) may be present in plants under special conditions. The relative importance of the various pathways depends on a number of factors species of plant, age of plant, part of plant examined, growth conditions, experimental conditions, and others. 

From my laboratory and the laboratories of Ef Racker, Frank Dickens and Melvin Calvin, the years that followed witnessed a series of parallel and often highly synergistic discoveries on the nature of the pentose phosphate pathway and the path of carbon in photosynthesis. Andrew Benson and others in Calvin s laboratory, had shown that phosphate esters of ribulose and sedoheptulose were early products of CO2 fixation in photosynthesis,and the immediate precursor of phosphoglyceric acid, and therefore the primary CO2 acceptor, appeared to be ribulose diphosphate. The major problems became (1) to find the enzyme or enzymes that catalyzed the formation of phosphoglyceric acid from ribulose diphosphate and (2) to define the reactions leading to the synthesis of ribulose diphosphate from triose and hexose phosphates. 

The isotope data of Bassham el al. (36) strongly support the in vivo operation of the ribulose diphosphate cycle. In short-term photosynthesis PGA labeled in carbon atoms 1 would give rise to 3,4-labeled F-6-P. This would yield S-7-P and pentose phosphate labeled as is shown in Scheme 2. S-7-P would be expected to be labeled equally in carbon atoms 3, 4, and 5 and this is the result obtained by Bassham et al. (36) 5 seconds after the introduction of C Oj. The low activity of C-4, relative to C-3 and C-5, observed by these workers after 0.4 second in C K)2, is not explained by the known reactions, since asymmetric labeling in hexose (C-4>C-3) has not been observed by them. Furthermore, both C-3 and C-4 of S-7-P arise from C-3 of hexose (represented by the symbol +) and should always be equally labeled. ... 

The conversion of pentoses to hexoses by the reversal of the cycle constitutes, in essence, the pathway of carbon in photosynthesis of green plants. The primary acceptor of CO2, ribulose diphosphate, is regenerated according to that scheme (cf. Chapt. XVI ). 

Formation of Ribulose Diphosphate. Up to this point the situation has remained relatively simple. But now ribulose diphosphate has to be regenerated so that it may accept another CO 2. Otherwise, photosynthesis would soon come to a halt. The pathway is analogous to the regeneration of hexose by the reverse of the pentose phosphate cycle. Here is a simplified schematic diagram ... 

The long delay in increase in activity of the two latter enzymes suggested that these changes might result from events not directly related to the initial illumination of etiolated leaves but perhaps to attainment of photosynthetic capacity by the leaf. This possibility appears to be confirmed by investigations in which -chlorophenyl-methylurea (CMU), a potent inhibitor of photosynthesis, was administered to etiolated leaves prior to illumination. The change in activity of ribulose-diphosphate carboxylase described above was not different from normal in CMU-treated leaves, but the usual increase in the level of the isomerase failed to occur. 

To summarize these observations First, the enzymes of this segment of the photosynthetic carbon reduction cycle are not controlled co-ordinately a single inductive step does not appear to affect the activity (and presumably the production) of these three enzymes in the same way. Second, the kinds of control mechanisms which appear to occur here are (1) a direct and prompt effect of illumination, reflected in the rapid increase in ribulose-diphosphate carboxylase activity, and (2) a more indirect kind of control, possibly involving induction of the other enzymes (e.g., ribose-phosphate isomerase) by small molecules produced in photosynthesis. Unfortunately, it has not been possible to demonstrate an increase in the level of either the isomerase or kinase by administration of glucose and some other carbohydrates to etiolated leaves in darkness. 

By contrast, the Calvin cycle in photosynthesis illustrates a positive feed-back mechanism. The concentration of Calvin cycle intermediates persisting in photosynthetic cells in the dark is very low. When photosynthesis commences the availability of ribulose diphosphate (RuDP) may limit the rate of COj assimilation. However, the formation of phosphoglyceric acid (PGA) immediately raises the level of other intermediates in the cycle, including that of RuDP, and this in turn speeds up the rate of CO assimilation. Similarly, when cells... 

Photosynthesis. The formation of carbohydrates in green plants by the process of photosynthesis is described in ihc entry on Photosynthesis. The synthetic mechanism involves the addition of carbon dioxide to ribulose-1,5-diphosphate and the subsequent formation of two molecules of 3-phosphoglyccric acid which are reduced to glyceraldehyde-3-phosphate. The triose phosphates are utilized to again from ribulose-5-phosphates by enzymes of the pentose phosphate cycle Phosphorylation or ribulose-5-phosphate with ATP regenerates ribulose-1.5-diphosphate to accept another molecule of carbon dioxide. See also Phosphorylation (Photosynthetlc). 

The Kolbe-Schmitt reaction is related to enzymatic carboxylations as of D-ribulose 1,5-diphosphate with carbon dioxide, a key step in photosynthesis (Section 20-9). The overall result is C—C bond formation by addition of C02 to an enolate salt or its enamine equivalent. 

In those organisms that perform photosynthesis, further phosphorylation of D-ribulose 5-phos-phate into D-ribulose 1,5-diphosphate by phosphoribulokinase represents an important prerequisite in CO2 fixation. As has already been mentioned, the key and extensively studied enzyme involved in this reaction is D-ribulose 1,5-bisphosphate carboxylase/oxygenase. The subfamily divergence in this multigene family has been studied in certain plants of Triticeae and other families [13]. The occurrence of this enzyme in anoxic Archaea is very interesting, since it had to evolve in the absence of molecular oxygen [14]. 

In subaerial C3 plants substrate and reactant (s and r, respectively, in Fig. 5.56) for photosynthesis are both gaseous (atmospheric) C02, which flows through the Calvin cycle (the dark reactions of photosynthesis see Box 1.10) to yield simple carbohydrates (p), which are in turn the source of various metabolic intermediates. The source of the intracellular (kinetic) isotopic fractionation during C fixation is the enzyme rubisco (D-ribulose 1,5-diphosphate carboxylase/oxygenase). There is also an isotopic fractionation resulting from the passage of C02 into the cell. Passive diffusion of C02, at a rate , favours 12C, but the fractionation is small... 

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.

After a time, several other sugar phosphates were identified. Most important among these were the seven-carbon compounds, sedoheptulose-7-phosphate (IX) and sedoheptulose-l,7-diphosphate (SDP) (X), and the five-carbon compounds, ribulose-l,5-diphos-phate (RuDP) (II) and ribose-5-phosphate (XI), xylulose-5-phosphate (XII), and ribulose-5-phosphate (I) (Benson, et al., 1952). The roles of these compounds in the path of carbon in photosynthesis became more clear after they had been degraded to locate the position of radiocarbon atoms within the individual molecules (Bassham et al., 1954). 

The evidence for the participation of D-ribulose 1,5-diphosphate as a C02-acceptor in photosynthesis came from several sources. First, the data... 

Fig. 3. The role of the pentose phosphate pathway in photosynthesis. One-sixth of the fructose 1,6-diphosphate formed is assimilated and five-sixths is converted back to ribulose l,S-diphosphate.

Malic acid is a very active intermediary product of grape metabolism. The vine contains the L-(—) malic isomer. The vine assimilates carbon dioxide in the air by a C3 mechanism (Ruflher et al., 1983). In this manner, during the dark phase of photosynthesis, the leaves and young green grapes fix CO2 on ribulose 1,5-diphosphate to produce phospho-glyceric acid, which condenses to form hexoses and may also become dehydrated into phosphoenol pyruvic acid. CO2, catalyzed by PEP carboxylase, is fixed on this acid to form oxaloacetic acid, which is, in turn, reduced into malic acid. 

What holds true for the structural elements of the plastids need not be valid for all of the enzyme systems of photosynthesis. A key enzyme in the secondary processes of photosynthesis is carboxydismutase which fixes CO2 into ribulose-1, 5-diphosphate (page 51). The enzyme is already present before the light-dependent differentiation of the plastid structure, at least in rye seedlings. Nonetheless, illumination induces an... 

Ribulose is the ketose corresponding to ribose. In general, the ketoses are designated by the ending -ulose (unless they have trivial names, such as fructose). Xylulose is an epimer to ribulose at C-3. Ribulose phosphate and diphosphate play a role in biologic intereonversions of the sugars and in photosynthesis (Chapt. XVI-4). 

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