Photosynthetic Carbohydrate Synthesis:- Carbon Dioxide Assimilation Occurs in Three Stages
The first stage in the assimilation of CO2 into biomolecules is the carbon-fixation reaction: condensation of CO2 with a five-carbon acceptor, ribulose 1,5-bisphosphate, to form two molecules of 3 phosphoglycerate. In the second stage, the 3-phos phoglycerate is reduced to triose phosphates. Overall, three molecules of CO2 are fixed to three molecules of ribulose 1,5-bisphosphate to form six molecules of glyceraldehyde 3-phosphate (18 carbons) in equilibrium with dihydroxyacetone phosphate. In the third stage, five of the six molecules of triose phosphate (15 car bons) are used to regenerate three molecules of ribulose 1,5-bisphosphate (15 carbons), the starting mate rial. The sixth molecule of triose phosphate, the net product of photosynthesis, can be used to make hexoses for fuel and building materials, sucrose for trans port to nonphotosynthetic tissues, or starch for storage. Thus, the overall process is cyclical, with the continuous conversion of CO2 to triose and hexose phosphates. Fructose 6-phosphate is a key intermediate in stage 3 of CO2 assimilation; it stands at a branch point, leading either to regeneration of ribulose 1,5-bisphosphate or to synthesis of starch. The pathway from hexose phosphate to pentose bisphosphate involves many of the same reactions used in animal cells for the conversion of pentose phosphates to hexose phosphates during the nonoxidative phase of the pentose phosphate pathway. In the photosynthetic assimilation of CO2, essentially the same set of reactions operates in the other direction, converting hexose phosphates to pentose phosphates. This reductive pentose phosphate cycle uses the same enzymes as the oxidative pathway, and several more enzymes that make the reductive cycle irreversible. All 13 enzymes of the path way are in the chloroplast stroma.
FIGURE 1 Plastids: their origins and interconversions. All types of plastids are bounded by a double membrane, and some (notably the mature chloroplast) have extensive internal membranes. The in ternal membranes can be lost (when a mature chloroplast becomes a proplastid) and resynthesized (as a proplastid gives rise to a pregranal plastid and then a mature chloroplast). Proplastids in nonphotosyn thetic tissues (such as root) give rise to amyloplasts, which contain large quantities of starch. All plant cells have plastids, and these organelles are the site of other important processes, including the syn thesis of essential amino acids, thiamine, pyridoxal phosphate, flavins, and vitamins A, C, E, and K.
FIGURE 2 The three stages of CO2 assimilation in photosynthetic organisms. Stoichiometries of three key intermediates (numbers in parentheses) reveal the fate of carbon atoms entering and leaving the cycle. As shown here, three CO2 are fixed for the net synthesis of one molecule of glyceraldehyde 3-phosphate. This cycle is the photosynthetic carbon reduction cycle, or the Calvin cycle.
Stage 1: Fixation of CO2 into 3-Phosphoglycerate An important clue to the nature of the CO2-assimilation mechanisms in photosynthetic organisms came in the late 1940s. Calvin and his associates illuminated a suspension of green algae in the presence of radioactive car bon dioxide (14CO2) for just a few seconds, then quickly killed the cells, extracted their contents, and with the help of chromatographic methods searched for the metabolites in which the labeled carbon first appeared. The first compound that became labeled was 3-phos phoglycerate, with the 14C predominantly located in the carboxyl carbon atom. These experiments strongly suggested that 3-phosphoglycerate is an early intermediate in photosynthesis. The many plants in which this three-carbon compound is the first intermediate are called C3 plants, in contrast with the C4 plants described below.
The enzyme that catalyzes incorporation of CO2 into an organic form is ribulose 1,5-bisphosphate carboxylase/oxygenase, a name mercifully shortened to rubisco. As a carboxylase, rubisco catalyzes the covalent attachment of CO2 to the five-carbon sugar ribulose 1,5 bisphosphate and cleavage of the unstable six-carbon intermediate to form two molecules of 3-phosphoglycerate, one of which bears the carbon introduced as CO2 in its carboxyl group (Fig. 2). The enzyme’s oxygenase activity is discussed in Section 20.2.
Plant rubisco, the crucial enzyme in the production of biomass from CO2, has a complex structure (Fig. 3a), with eight identical large subunits (Mr 53,000; encoded in the chloroplast genome, or plastome), each containing a catalytic site, and eight identical small sub units (Mr 14,000; encoded in the nuclear genome) of uncertain function. The rubisco of photosynthetic bacteria is simpler in structure, having two subunits that in many respects resemble the large subunits of the plant enzyme (Fig. 3b). This similarity is consistent with the endosymbiont hypothesis for the origin of chloroplasts . The plant enzyme has an exceptionally low turnover number; only three molecules of CO2 are fixed per second per molecule of rubisco at 25 C. To achieve high rates of CO2 fixation, plants therefore need large amounts of this enzyme. In fact, rubisco makes up almost 50% of soluble protein in chloroplasts and is prob ably one of the most abundant enzymes in the biosphere. Central to the proposed mechanism for plant rubisco is a carbamoylated Lys side chain with a bound Mg2 ion. The Mg+2 ion brings together and orients the reactants at the active site and polarizes the CO2, opening it to nucleophilic attack by the five-carbon enediolate reaction intermediate formed on the enzyme (Fig. 5). The resulting six-carbon inter mediate breaks down to yield two molecules of 3 phosphoglycerate.
FIGURE 3 Structure of ribulose 1,5-bisphosphate carboxylase (rubisco). (a) Top and side view of a ribbon model of rubisco from spinach (PDB ID 8RUC). The enzyme has eight large subunits (blue) and eight small ones (gray), tightly packed into a structure of Mr 550,000. Rubisco is present at a concentration of about 250 mg/mL in the chloroplast stroma, corresponding to an extraordinarily high concentration of active sites (~4 mM). Amino acid residues of the active site are shown in yellow, Mg2+ in green. (b) Ribbon model of rubisco from the bacterium Rhodospirillum rubrum (PDB ID 9RUB). The subunits are in gray and blue. A Lys residue at the active site that is carboxylated to a carbamate in the active enzyme is shown in red. The substrate, ribulose 1,5-bisphosphate, is yellow; Mg2+ is green.
FIGURE 4 Central role of Mg2+ in the catalytic mechanism of rubisco. (Derived from PDB ID 1RXO) Mg2+ is coordinated in a roughly octahedral complex with six oxygen atoms: one oxygen in the carbamate on Lys201; two in the carboxyl groups of Glu204 and Asp203; two at C-2 and C-3 of the substrate, ribulose 1,5-bisphosphate; and one in the other substrate, CO2. A water molecule occupies the Co2–binding site in this crystal structure. (Residue numbers refer to the spinach enzyme.)
MECHANISM FIGURE 5 First stage of CO2assimilation: rubisco’s carboxylase activity. The CO2-fixation reaction is catalyzed by ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco). 1 Ribulose 1,5 bisphosphate forms an enediolate at the active site. 2CO2, polarized by the proximity of the Mg2 ion, undergoes nucleophilic attack by the enediolate, producing a branched six-carbon sugar. 3Hydroxy lation at C-3 of this sugar, followed by aldol cleavage 4, forms one molecule of 3-phosphoglycerate, which leaves the enzyme active site.
5 The carbanion of the remaining three-carbon fragment is protonated by the nearby side chain of Lys175, generating a second molecule of 3-phosphoglycerate. The overall reaction therefore accomplishes the combination of one CO2 and one ribulose 1,5-bisphosphate to form two molecules of 3-phosphoglycerate, one of which contains the carbon atom from CO2 (red).
FIGURE 6 Role of rubisco activase in the carbamoylation of Lys201 of rubisco. When the substrate ribulose 1,5-bisphosphate is bound to the active site, Lys201 is not accessible. Rubisco activase couples ATP hydrolysis to expulsion of the bound sugar bisphosphate, exposing Lys201; this Lys residue can now be carbamoylated with CO2 in a re action that is apparently not enzyme-mediated. Mg+2 is attracted to and binds to the negatively charged carbamoyl-Lys, and the enzyme is thus activated.
As the catalyst for the first step of photosynthetic CO2 assimilation, rubisco is a prime target for regulation. The enzyme is inactive until carbamoylated on the amino group of Lys201 (Fig. 7). Ribulose 1,5 bisphosphate inhibits carbamoylation by binding tightly to the active site and locking the enzyme in the “closed” conformation, in which Lys201 is inaccessible. Rubisco activase overcomes the inhibition by promoting ATP dependent release of the ribulose 1,5-bisphosphate, ex posing the Lys amino group to nonenzymatic carbamoylation by CO2; this is followed by Mg2 binding, which activates the rubisco. Rubisco activase in some species is activated by light through a redox mechanism . Another regulatory mechanism involves the “nocturnal inhibitor” 2-carboxyarabinitol 1-phosphate, a naturally occurring transition-state analog (see Box 6–3) with a structure similar to that of the -keto acid intermediate of the rubisco reaction . This compound, synthesized in the dark in some plants, is a potent inhibitor of carbamoylated rubisco. It is either broken down when light returns or is expelled by rubisco activase, activating the rubisco.
Stage 2: Conversion of 3-Phosphoglycerate to Glyceraldehyde 3-Phosphate The 3-phosphoglycerate formed in stage 1 is converted to glyceraldehyde 3-phosphate in two steps that are essentially the reversal of the corresponding steps in glycolysis, with one exception: the nucleotide cofactor for the reduction of 1,3-bisphosphoglycerate is NADPH rather than NADH (Fig. 7). The chloroplast stroma contains all the glycolytic enzymes except phos phoglycerate mutase. The stromal and cytosolic enzymes are isozymes; both sets of enzymes catalyze the same reactions, but they are the products of different genes.
In the first step of stage 2, the stromal 3-phospho glycerate kinase catalyzes the transfer of a phosphoryl group from ATP to 3-phosphoglycerate, yielding 1,3-bisphosphoglycerate. Next, NADPH donates electrons in a reduction catalyzed by the chloroplast-specific isozyme of glyceraldehyde 3-phosphate dehydroge nase, producing glyceraldehyde 3-phosphate and Pi. Triose phosphate isomerase then interconverts glycer aldehyde 3-phosphate and dihydroxyacetone phosphate.
FIGURE 7 Second stage of CO2 assimilation. 3-Phosphoglycerate is converted to glyceraldehyde 3-phosphate (red arrows). Also shown are the alternative fates of the fixed carbon of glyceraldehyde 3-phosphate (blue arrows). Most of the glyceraldehyde 3-phosphate is recycled to ribulose 1,5-bisphosphate as shown in Figure 8. A small fraction of the “extra” glyceraldehyde 3-phosphate may be used immediately as a source of energy, but most is converted to sucrose for transport or is stored in the chloroplast as starch. In the latter case, glyceraldehyde 3-phosphate condenses with dihydroxyacetone phosphate in the stroma to form fructose 1,6-bisphosphate, a precursor of starch. In other situations, the glyceraldehyde 3-phosphate is converted to dihydroxyacetone phosphate, which leaves the chloroplast via a specific transporter and, in the cytosol, can be degraded glycolytically to provide energy or used to form fructose 6-phosphate and hence sucrose.
Most of the triose phosphate thus produced is used to regenerate ribulose 1,5-bisphosphate; the rest is either converted to starch in the chloroplast and stored for later use or immediately exported to the cytosol and con verted to sucrose for transport to growing regions of the plant. In developing leaves, a significant portion of the triose phosphate may be degraded by glycolysis to provide energy.
Stage 3: Regeneration of Ribulose 1,5-Bisphosphate from Triose Phosphates The first reaction in the assimilation of CO2 into triose phosphates consumes ribulose 1,5-bisphosphate and, for continuous flow of CO2 into carbohydrate, ribulose 1,5-bisphosphate must be constantly regenerated. This is accomplished in a series of reactions (Fig. 8) that, together with stages 1 and 2, constitute the cyclic pathway shown in Figure 20–4. The product of the first assimilation reaction (3-phosphoglycerate) thus undergoes transformations that regenerate ribulose 1,5-bisphosphate. The intermediates in this path way include three-, four-, five-, six-, and seven-carbon sugars. In the following discussion, all step numbers re fer to Figure 20–10. Steps 1 and 4 are catalyzed by the same enzyme, transaldolase. It first catalyzes the reversible condensation of glyceraldehyde 3-phosphate with dihydroxy acetone phosphate, yielding fructose 1,6-bisphosphate (step 1); this is cleaved to fructose 6-phosphate and Pi by fructose 1,6-bisphosphatase (FBPase-1) in step 2. The reaction is strongly exergonic and essentially irreversible. Step 3 is catalyzed by transketolase, which contains thiamine pyrophosphate (TPP) as its prosthetic group (see Fig. 14–13a) and requires Mg2+
FIGURE 8 Third stage of CO2 assimilation. This schematic diagram shows the interconversions of triose phosphates and pentose phosphates. Black dots represent the number of carbons in each compound. The starting materials are glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Reactions catalyzed by transaldolase (1 and 4) and transketolase (3 and 6) produce pentose phosphates that are converted to ribulose 1,5-bisphosphate—ribose 5-phosphate by ribose 5-phosphate isomerase (7) and xylulose 5-phosphate by ribulose 5-phosphate epimerase (8). In step 9, ribulose 5-phosphate is phosphorylated, regenerating ribulose 1,5 bisphosphate. The steps with blue arrows are exergonic and make the whole process irreversible: steps 2 fructose 1,6-bisphosphatase, 5 sedoheptulose bisphosphatase, and 9 ribulose 5-phosphate kinase.
FIGURE 9 Transketolase-catalyzed reactions of the Calvin cycle. (a)General reaction catalyzed by transketolase: the transfer of a two-carbon group, carried temporarily on enzyme bound TPP, from a ketose donor to an aldose acceptor. (b)Conversion of a hexose and a triose to a four-carbon and a five-carbon sugar (step 3 of Fig. 20–10). (c)Conversion of seven carbon and three-carbon sugars to two pentoses (step 6 of Fig. 8).
Transketolase catalyzes the reversible transfer of a 2-carbon ketol group (CH2OHOCOO) from a ketose phosphate donor, fructose 6-phosphate, to an aldose phosphate acceptor, glyceraldehyde 3-phosphate (Fig. 20–11a, b), forming the pentose xylulose 5-phosphate and the tetrose erythrose 4-phosphate. In step 4, transaldolase acts again, combining erythrose 4-phosphate with dihydroxyacetone phosphate to form the seven-carbon sedoheptulose 1,7-bisphosphate. An enzyme unique to plastids, sedoheptulose 1,7-bisphosphatase, converts the bisphosphate to sedoheptulose 7-phosphate (step 5); this is the second irreversible reaction in the pathway. Transketolase now acts again, converting sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate to two pentose phosphates in step 6 (Fig. 8c). Figure 20–12shows how a two-carbon fragment is temporarily carried on the transketolase cofactor TPP and condensed with the three carbons of glyceraldehyde 3-phosphate in step 6. The pentose phosphates formed in the transketolase reactions—ribose 5-phosphate and xylulose 5-phosphate—are converted to ribulose 5-phosphate (steps 7 and 8), which in the final step (9) of the cycle is phosphorylated to ribulose 1,5-bisphosphate by ribulose 5-phosphate kinase (Fig. 10). This is the third very exergonic reaction of the pathway, as the phosphate an hydride bond in ATP is swapped for a phosphate ester in ribulose 1,5-bisphosphate.
FIGURE 10 TPP as a cofactor for transketolase. Transketolase transfers a two-carbon group from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate, producing two pentose phosphates . Thiamine pyrophosphate serves as a temporary carrier of the two-carbon unit and as an electron sink to facilitate the reactions.
