Carbon Fixation

All oxygenic organisms from the simplest prokaryotic cyanobacteria to the most complicated land plants reduce atmospheric CO2 to sugar phosphates by a pathway referred to as the Calvin cycle or RPP pathway. This pathway located in chloroplasts is the primary carboxylating mechanism in plants and is comprised of thirteen reactions catalyzed by eleven enzymes. The pathway has four principal features which include : (i) carboxylation, in which CO2 is joined to the acceptor RuBP to form two molecules of 3-PGA, (ii) reduction, in which 3-PGA is reduced to triose-P at the expense of ATP and NADPH, (iii) regeneration, in which five molecules of triose-P are rearranged to yield three molecules of pentose phosphate in the "sugar phosphate" shuttle, (iv) autocatalysis i.e, the cycle acts as an autocatalytic breeder reaction in which one molecule of triose-P is generated from three molecules of CO2 for every three turns of the cycle. Triose-P thus generated may either be utilized in the synthesis of starch or sucrose or may re-enter the cycle to form more of primary acceptor RuBP. In the second case, the cycle generates more CO2 acceptor than it consumes.

2.1. Reactions of Calvin cycle

The delineated path of carbon in the Calvin cycle has remained virtually unchanged from that originally outlined by Melvin Calvin in late 1950's (15). Subsequent studies spanning about over 30 years have remained restricted mainly to the detailed characteristics of enzymes including their regulation. In the last decade, however, much emphasis has been given to their molecular properties including gene regulation (6-12). The main reactions of the pathway are shown in Fig. 1.


The key reaction of the Calvin cycle is the binding of atmospheric CO2 to the acceptor RuBP to form two molecules of 3-PGA. The reaction is highly exergonic (F'= -8.4Kcal) and is catalyzed by the enzyme Rubisco. This carboxylation reaction is based on an enediol mechanism involving five steps (13, 14). Oxygen competes with CO2, giving to the alternative ribulose-1,5-bisphosphate oxygenase reaction, yielding one molecule each of 3-PGA and 2-phosphoglycolate. Since CO2 and O2 compete for the same site of the enzyme, the rates of the two reactions are determined by the concentrations of the two gases. Factors such as pH and temperature also affect the rates of these two reactions. Under atmospheric conditions (250 pM O2; 10 nM

Rubisco Mechanism

Fig. 1. The autocatalytic Calvin cycle showing three distinct phases of the cycle. 1, Rubisco; 2, 3-PGA kinase; 3, Glyceraldehyde-3-P dehydrogenase; 4, Triose phosphate isomerase; 5, Aldolase; 6, FBPase; 7, Transketolase; 8, Aldolase; 9, SBPase; 10, Transketolase; 11, Ribulose-5-P epimerase; 12, Ribose-5-P isomerase; 13, Phosphoribulokinase.

Fig. 1. The autocatalytic Calvin cycle showing three distinct phases of the cycle. 1, Rubisco; 2, 3-PGA kinase; 3, Glyceraldehyde-3-P dehydrogenase; 4, Triose phosphate isomerase; 5, Aldolase; 6, FBPase; 7, Transketolase; 8, Aldolase; 9, SBPase; 10, Transketolase; 11, Ribulose-5-P epimerase; 12, Ribose-5-P isomerase; 13, Phosphoribulokinase.

CO2), carboxylase activity is 3 to 5 times that of oxygenase activity. However, as the temperature rises, the CO2/O2 specificity decreases, and as a consequence the ratio of oxygenation to carboxylation increases.

Catalysis of the carboxylation of RuBP by Rubisco is a very slow process; the turnover number of each subunit amounts to 3.3 per second. Because of this low turnover number, very large amounts of enzyme are needed to catalyze fluxes required for photosynthesis. That may be the reason why Rubisco amounts to 50% of the total soluble proteins in leaves. The enzyme Rubisco is the best studied plant enzyme and has attracted much attention in the past (15-20). Despite the relative abundance of Rubisco in leaves, the reaction catalyzed by the enzyme is regulatory. In vivo the enzyme is activated by light which brings favourable changes in pH, Mg2+ and the metabolites in the stromal compartment. Another mechanism of Rubisco activation involves a nuclear encoded chloroplast protein, referred to as Rubisco activase which catalyzes an ATP dependent activation of the inactive E-RuBP complex to the active E-AC02-Mg2+-RuBP complex (21). 2-Carboxy arabinitol-1-phosphate (CA1P) an analogue of the intermediate of the Rubisco reaction is a naturally occurring inhibitor of Rubisco, which accumulates in the dark and low light conditions and binds to the activated form of the enzyme (19). Xylulose-bisphosphate, another substrate analogue is the second natural inhibitor of the enzyme, which binds tightly at the active site (22). Reduction

The 3-PGA formed in carboxylation reaction is phosphorylated by 3-PGA kinase to 1,3-bisphosphoglycerate (BPGA) using ATP synthesized during light reaction. In this reaction, a mixed anhydride is formed between the new phosphate residue and the carboxyl group. The reaction has large +ve F' value of about 4.5 Kcal, indicating that the equilibrium position of this reaction lies towards the formation of PGA. Hence for the reaction to operate in the direction of BPGA synthesis, a high ratio of the substrate to product ([PGA] [ATP])/([BPGA] [ADP]) is required. This situation does exist in chloroplast where concentration of 3-PGA is much high relative to that of BPGA resulting in very high ratios of 3-PGA/BPGA. The enzyme 3-PGA kinase is a monomer with molecular mass of about 38 KDa. The plant has two isoenzymes located in chloroplast and cytosol of leaf tissues (23). The chloroplastic enzyme accounting for about 90% of the total leaf activity is inhibited by both AMP and ADP.

The reduction of BPGA to D-glyceraldehyde-3-phosphate (GAP) is catalyzed by the enzyme NADP:glyceraldehyde-3-P dehydrogenase (GAPDH) located exclusively in the chloroplasts. The formation of GAP is only reductive step in the pathway and is also the major site of Pi release. Since the F' value for the reaction is -ve (-1.5 Kcal), the equilibrium position of this reaction lies in the direction of GAP formation. The chloroplastic form of this enzyme is an oligomer with two different kinds of subunits named as A and B with respective molecular mass of 37 and 43 KDa (24). Homotetrameric A4 form accounts for about 20% of the chloroplastic activity (25). Besides this, chloroplastic enzyme has also been shown to occur in various other oligomeric forms such as A2B2 (160 KDa), A4B4 (300 KDa) and A8B8 (600 KDa). The enzyme is light activated by thioredoxin system as well as by different metabolites (26, 27).

The interconversion of GAP to DHAP is catalyzed by the enzyme triose phosphate isomerase (TPI). The reaction proceeds via an 1,2-enediol intermediate and is readily reversible (F'= -1.8 Kcal). At equilibrium, the ratio of DHAP to GAP ia about 22:1. The enzyme TPI is a homodimer with a subunit molecular mass of about 27 KDa (28). The enzyme is completely inhibited by RuBP, Pi, fru-1,6-bisphosphate (fru-l,6-P2) and 3-PGA. 2-phosphoglycolate is the stronger inhibitor of the enzyme (29).


This phase of Calvin cycle consisting of two parts generates RuBP (CO2 acceptor) from trio se-phosphates. In the first part, fructose-1,6-bisphosphatase (FBPase), sedoheptulose-l,7-bisphosphatase (SBPase), transketolase and aldolase convert five C-3 units (triose-P) into three C-5 units. In the second part, pentose phosphates are converted to RuBP in reactions catalyzed by phosphoribose isomerase, ribulose-5-phosphate 3-epimerase and ribulose-5-phosphate kinase. In the first reaction of this phase catalyzed by the enzyme aldolase, the two triose-Ps i.e. GAP and DHAP condense in a reversible manner to fru-l,6-P2- There are two aldolase reactions in the Calvin cycle, and it is believed that both the reactions are catalyzed by the single enzyme. The F' for the above reaction is highly negative (-5.5 Kcal), indicating that the equilibrium for this reaction lies towards formation of fru-l,6-P2. However, the reaction has two substrates and one product. The equilibrium position is greatly influenced by the concentrations of the components involved. The enzyme aldolase from chloroplast is a tetramer of 148 KDa, comprising subunits of 38 KDa (30). The enzyme is inhibited by RuBP, ADP and 3-PGA.

Fru-1,6-P2 formed as above is hydrolyzed by FBPase to yield fructose-6-P (fru-6-P) and Pi. This is second Pi releasing step in Calvin cycle. The reaction has large - F (-4.0 Kcal), indicating formation of fru-6-P to be favoured and suggesting the enzyme to be regulatory. The enzyme FBPase has been studied extensively as it plays a key role in both the Calvin cycle in chloroplast and the sucrose synthesis in cytosol. Accordingly, two isoenzymes have been identified in photosynthetic tissues both of which are regulatory in their respective pathways(31). Significant differences have been shown to exist in sequences of chloroplastic and cytosolic enzymes from different sources (32). The chloroplastic enzyme is a homo tetramer with a molecular mass of 170 KDa. The enzyme is largely specific for fru-l,6-P2 and catalyzes an essentially irreversible reaction requiring divalent cation (Mg2+) for activity. The enzyme is active in light and almost inactive in dark.

The next reaction is catalyzed by transketolase which transfers a carbohydrate residue with two carbon atoms from fru-6-P to GAP, yielding xylulose-5-P (Xu5P) and erythrose-4-P (E4P) in a reversible manner (F-1.47 Kcal). The enzyme uses thiamine pyrophosphate (TPP) as the coenzyme. Like aldolase reactions, there are two transketolase reactions in the Calvin cycle. Both are again thought to be catalyzed by the single enzyme. In the second aldol condensation reaction, E4P joins DHAP to yield sedoheptulose-l,7-P2 (SBP), which is subsequently hydrolyzed by the enzyme SBPase. This reaction ( F'=-4.0 Kcal) is similar to the hydrolysis of fru-l,6-P2, although the two reactions are catalyzed by two different enzymes. The enzyme

SBPase is a dimer comprising of two identical subunits with molecular mass of 35 KDa each (33). Though the enzyme resembles FBPase in many kinetic and regulatory properties, it is quite distinct from FBPase and immunologically unrelated (34). Like FBPase, SBPase is also activated by light. SBPase activity is also regulated by stromal pH and Mg2+ levels both of which change in response to light/dark transitions. Besides, products of SBPase reaction also exert an additional control on the enzyme activity (34).

In the next step, the second transketolase reaction transfers a C2 unit from sedoheptulose-7-P as before to GAP to produce Xu5P and ribose-5-P. Xu5P in turn is converted to ribulose-5-P by the enzyme ribulose-5-P, 3-epimerase. This reaction proceeds via keto-enol isomerization with 3,3-enediol as intermediary product. The enzyme has not been well characterized from plant sources and is a homodimer with a subunit molecular mass of 23 KDa. The conversion of ribose-5-P to ribulose-5-P is catalyzed by ribose-5-P isomerase, again via an enediol as intermediate, although in 1,2 position. The reaction is again freely reversible. The enzyme is a homodimer with subunit molecular mass of 26 KDa. The three molecules of ribulose-5-P formed as above are converted to the CC>2 acceptor RuBP with consumption of ATP by phosphoribulo kinase (PRK). This reaction utilizes one-third of the ATP required for CO2 fixation and favours the formation of RuBP with F' value of -5.2 Kcal. The purified enzyme is a homodimer comprising of two identical subunits of 42-44 KDa (35, 36). The enzyme is regulated by light. The light activated enzyme is competitively inhibited by 6-phosphogluconate, RuBP and Pi (37). The thirteen enzymatic reactions of Calvin cycle discussed above bring about reduction of atmospheric CO2 and regenerate the acceptor molecule. The overall reaction can be written as

6CO2+18ATP+12NADPH+12H++11H20—► Fru-6-P+ 18ADP+12NADP+ 17Pi

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  • sabrina
    Which enzyme cnvert DHAP to fru1,6 bsphosphate in calvin cycle?
    4 years ago
  • patrick
    How is starch synthesized from co2 in plant?
    1 year ago

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