The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water

II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
We saw in Chapter 16 that glucose could be formed from noncarbohydrate precursors, such as lactate and amino acids,
by gluconeogenesis. The synthesis of glucose from these compounds is simplified because the carbons are already
incorporated into relatively complex organic molecules. In contrast, the source of the carbon atoms in the Calvin cycle is
the simple molecule carbon dioxide. In this extremely important process, carbon dioxide gas is trapped in a form that is
useful for many processes. The Calvin cycle brings into living systems the carbon atoms that will become constituents of
nucleic acids, proteins, and fats. Photosynthetic organisms are called autotrophs (literally "self-feeders") because they
can synthesize glucose from carbon dioxide and water, by using sunlight as an energy source, and then recover some of
this energy from the synthesized glucose through the glycolytic pathway and aerobic metabolism. Organisms that obtain
energy from chemical fuels only are called heterotrophs, which ultimately depend on autotrophs for their fuel. The
Calvin cycle also differs from gluconeogenesis in where it takes place in photosynthetic eukaryotes. Whereas
gluconeogenesis takes place in the cytoplasm, the Calvin cycle takes place in the stroma of chloroplasts, the
photosynthetic organelles.
The Calvin cycle comprises three stages (Figure 20.1):
1. The fixation of CO2 by ribulose 1,5-bisphosphate to form two molecules of 3-phosphoglycerate.
2. The reduction of 3-phosphoglycerate to form hexose sugars.
3. The regeneration of ribulose 1,5-bisphosphate so that more CO2 can be fixed.
Although we will focus on the Calvin cycle, other means of fixing carbon dioxide into hexose sugars exist in the
photosynthetic world, notably a version of the citric acid cycle running in reverse.
20.1.1. Carbon Dioxide Reacts with Ribulose 1,5-bisphosphate to Form Two Molecules
of 3-Phosphoglycerate
The first step in the Calvin cycle is the fixation of CO2. The CO2 molecule condenses with ribulose 1,5-bisphosphate to
form an unstable six-carbon compound, which is rapidly hydrolyzed to two molecules of 3-phosphoglycerate.
The initial incorporation of CO2 into 3-phosphoglycerate was revealed through the use of a carbon-14 radioactive tracer
(Figure 20.2). This highly exergonic reaction [∆ G°´ = 12.4 kcal mol 1 ( 51.9 kJ mol 1)] is catalyzed by ribulose 1,5bisphosphate carboxylase/oxygenase (usually called rubisco), an enzyme located on the stromal surface of the thylakoid
membranes of chloroplasts. This important reaction is the rate-limiting step in hexose synthesis. Rubisco in chloroplasts
consists of eight large (L, 55-kd) subunits and eight small (S, 13-kd) ones (Figure 20.3). Each L chain contains a
catalytic site and a regulatory site. The S chains enhance the catalytic activity of the L chains. This enzyme is very
abundant in chloroplasts, constituting more than 16% of their total protein. In fact, rubisco is the most abundant enzyme
and probably the most abundant protein in the biosphere. Large amounts are present because rubisco is a slow enzyme;
its maximal catalytic rate is only 3 s 1.
Rubisco requires a bound divalent metal ion for activity, usually magnesium ion. Like the zinc ion in the active site of
carbonic anhydrase (Section 9.2.1), this metal ion serves to activate a bound substrate molecule by stabilizing a negative
charge. Interestingly, a CO2 molecule other than the substrate is required to complete the assembly of the Mg2+ binding
site in rubisco. This CO2 molecule adds to the uncharged ε-amino group of lysine 201 to form a carbamate. This
negatively charged adduct then binds the Mg2+ ion. The formation of the carbamate is facilitated by the enzyme rubisco
activase, although it will also form spontaneously at a lower rate.
The metal center plays a key role in binding ribulose 1,5-bisphosphate and activating it so that it will react with CO2
(Figure 20.4). Ribulose 1,5-bisphosphate binds to Mg2+ through its keto group and an adjacent hydroxyl group. This
complex is readily deprotonated to form an enediolate intermediate. This reactive species, analogous to the zinchydroxide species in carbonic anhydrase (Section 9.2.2), couples with CO2, forming the new carbon-carbon bond. The
resulting product is coordinated to the Mg2+ ion through three groups, including the newly formed carboxylate. A
molecule of H2O is then added to this β-ketoacid to form an intermediate that cleaves to form two molecules of 3phosphoglycerate (Figure 20.5).
20.1.2. Catalytic Imperfection: Rubisco Also Catalyzes a Wasteful Oxygenase Reaction
The reactive intermediate generated on the Mg2+ ion sometimes reacts with O2 instead of CO2. Thus, rubisco also
catalyzes a deleterious oxygenase reaction. The products of this reaction are phosphoglycolate and 3-phosphoglycerate
(Figure 20.6). The rate of the carboxylase reaction is four times that of the oxygenase reaction under normal atmospheric
conditions at 25°C; the stromal concentration of CO2 is then 10 µM and that of O2 is 250 µM. The oxygenase reaction,
like the carboxylase reaction, requires that lysine 201 be in the carbamate form. Because this carbamate forms only in the
presence of CO2, this property would prevent rubisco from catalyzing the oxygenase reaction exclusively when CO2 is
absent.
Phosphoglycolate is not a versatile metabolite. A salvage pathway recovers part of its carbon skeleton (Figure 20.7). A
specific phosphatase converts phosphoglycolate into glycolate, which enters peroxisomes (also called microbodies;
Figure 20.8). Glycolate is then oxidized to glyoxylate by glycolate oxidase, an enzyme with a flavin mononucleotide
prosthetic group. The H2O2 produced in this reaction is cleaved by catalase to H2O and O2. Transamination of
glyoxylate then yields glycine. Two glycine molecules can be used to form serine, a potential precursor of glucose, with
the release of CO2 and ammonia (NH4 +). The ammonia, used in the synthesis of nitrogen-containing compounds, is
salvaged by glutamine synthetase reaction.
This salvage pathway serves to recycle three of the four carbon atoms of two molecules of glycolate. However, one
carbon atom is lost as CO2. This process is called photorespiration because O2 is consumed and CO2 is released.
Photorespiration is wasteful because organic carbon is converted into CO2 without the production of ATP, NADPH, or
another energy-rich metabolite. Moreover, the oxygenase activity increases more rapidly with temperature than the
carboxylase activity, presenting a problem for trop-ical plants (Section 20.2.3). Evolutionary processes have presumably
enhanced the preference of rubisco for carboxylation. For instance, the rubisco of higher plants is eightfold as specific
for carboxylation as that of photosynthetic bacteria.
20.1.3. Hexose Phosphates Are Made from Phosphoglycerate, and Ribulose 1,5bisphosphate Is Regenerated
The 3-phosphoglycerate product of rubisco is next converted into three forms of hexose phosphate: glucose 1-phosphate,
glucose 6-phosphate, and fructose 6-phosphate. Recall that these isomers are readily interconvertible (Sections 16.1.2
and 16.1.11). The steps in this conversion (Figure 20.9) are like those of the gluconeogenic pathway (Section 16.3.1),
except that glyceraldehyde 3-phosphate dehydrogenase in chloroplasts, which generates glyceraldehyde 3-phosphate
(GAP), is specific for NADPH rather than NADH. Alternatively, the glyceraldehyde 3-phosphate can be transported to
the cytosol for glucose synthesis. These reactions and that catalyzed by rubisco bring CO2 to the level of a hexose,
converting CO2 into a chemical fuel at the expense of NADPH and ATP generated from the light reactions.
The third phase of the Calvin cycle is the regeneration of ribulose 1,5-bisphosphate, the acceptor of CO2 in the first step.
The problem is to construct a five-carbon sugar from six-carbon and three-carbon sugars. A transketolase and an aldolase
play the major role in the rearrangement of the carbon atoms. The transketolase, which we will see again in the pentose
phosphate pathway (Section 20.2.3), requires the coenzyme thiamine pyrophosphate (TPP) to transfer a two-carbon unit
(CO-CH2OH) from a ketose to an aldose.
We will consider the mechanism of transketolase when we meet it again in the pentose phosphate pathway (Section
20.3.2). Aldolase, which we have already encountered in glycolysis (Section 16.1.3), catalyzes an aldol condensation
between dihydroxyacetone phosphate and an aldehyde. This enzyme is highly specific for dihydroxyacetone phosphate,
but it accepts a wide variety of aldehydes.
With these enzymes, the construction of the five-carbon sugar proceeds as shown in Figure 20.10.
Finally, ribose-5-phosphate is converted into ribulose 5-phosphate by phosphopentose isomerase while xylulose 5phosphate is converted into ribulose 5-phosphate by phosphopentose epimerase. Ribulose 5-phosphate is converted into
ribulose 1,5-bisphosphate through the action of phosphoribulose kinase (Figure 20.11). The sum of these reactions is
This series of reactions completes the Calvin cycle (Figure 20.12). The sum of all the reactions results in the generation
of a hexose and the regeneration of the starting compound, ribulose 5-phosphate. In essence, ribulose 1,5-bisphosphate
acts catalytically, similarly to oxaloacetate in the citric acid cycle.
20.1.4. Three Molecules of ATP and Two Molecules of NADPH Are Used to Bring
Carbon Dioxide to the Level of a Hexose
What is the energy expenditure for synthesizing a hexose? Six rounds of the Calvin cycle are required, because one
carbon atom is reduced in each round. Twelve molecules of ATP are expended in phosphorylating 12 molecules of 3phosphoglycerate to 1,3-bisphosphoglycerate, and 12 molecules of NADPH are consumed in reducing 12 molecules of
1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate. An additional six molecules of ATP are spent in regenerating
ribulose 1,5-bisphosphate. We can now write a balanced equation for the net reaction of the Calvin cycle.
Thus, three molecules of ATP and two molecules of NADPH are consumed in incorporating a single CO2 molecule into
a hexose such as glucose or fructose.
20.1.5. Starch and Sucrose Are the Major Carbohydrate Stores in Plants
Plants contain two major storage forms of sugar: starch and sucrose. Starch, like its animal counterpart glycogen, is a
polymer of glucose residues, but it is less branched than glycogen because it contains a smaller proportion of α-1,6glycosidic linkages (Section 11.2.2). Another difference is that ADP-glucose, not UDP-glucose, is the activated
precursor. Starch is synthesized and stored in chloroplasts.
In contrast, sucrose (common table sugar), a disaccharide, is synthesized in the cytosol. Plants lack the ability to
transport hexose phosphates across the chloroplast membrane, but an abundant phosphate translocator mediates the
transport of triose phosphates from chloroplasts to the cytosol in exchange for phosphate. Fructose 6-phosphate formed
from triose phosphates joins the glucose unit of UDP-glucose to form sucrose 6-phosphate (Figure 20.13). Hydrolysis of
the phosphate ester yields sucrose, a readily transportable and mobilizable sugar that is stored in many plant cells, as in
sugar beets and sugar cane.
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.1. Calvin Cycle. The Calvin cycle consists of three stages. Stage 1 is the fixation of carbon by the
carboxylation of ribulose 1,5-bisphosphate. Stage 2 is the reduction of the fixed carbon to begin the synthesis of hexose.
Stage 3 is the regeneration of the starting compound, ribulose 1,5-bisphosphate.
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.2. Tracing the Fate of Carbon Dioxide. Radioactivity from 14CO2 is incorporated into 3-phosphoglycerate
within 5 s in irradiated cultures of algae. After 60 s, the radioactivity appears in many compounds, the intermediates
within the Calvin cycle. [Courtesy of Dr. J. A. Bassham.]
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.3. Structure of Rubisco. The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) comprises
eight large subunits (one shown in red and the others in yellow) and eight small subunits (one shown in blue and
the others in white). The active sites lie in the large subunits.
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.4. Role of the Magnesium Ion in the Rubisco Mechanism. Ribulose 1,5-bisphosphate binds to a magnesium
ion that is linked to rubisco through a glutamate residue, an aspartate residue, and the lysine carbamate. The coordinated
ribulose 1,5-bisphosphate gives up a proton to form a reactive enediolate species that reacts with CO2 to form a new
carbon-carbon bond.
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.5. Formation of 3-Phosphoglycerate. The overall pathway for the conversion of ribulose 1,5 bisphosphate
and CO2 into two molecules of 3-phosphoglycerate. Although the free species are shown, these steps take place on the
magnesium ion.
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.6. A Wasteful Side Reaction. The reactive enediolate intermediate on rubisco also reacts with molecular
oxygen to form a hydroperoxide intermediate, which then proceeds to form one molecule of 3-phosphoglycerate and one
molecule of phosphoglycolate.
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.7. Photorespiratory Reactions. Phosphoglycolate is formed as a product of the oxygenase reaction in
chloroplasts. After dephosphorylation, glycolate is transported into peroxisomes where it is converted into glyoxylate
and then glycine. In mitochondria, two glycines are converted into serine, after losing a carbon as CO2 and ammonia.
The ammonia is salvaged in chloroplasts.
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.8. Electron Micrograph of a Peroxisome Nestled between Two Chloroplasts. [Courtesy of Dr. Sue Ellen
Frederick.]
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.9. Hexose Phosphate Formation. 3-Phosphoglycerate is converted into fructose 6-phosphate in a pathway
parallel to that of glyconeogenesis.
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.10. Formation of Five-Carbon Sugars. First, transketolase converts a six-carbon sugar and a three-carbon
sugar into a four-carbon sugar and a five-carbon sugar. Then, aldolase combines the four-carbon product and a threecarbon sugar to form a seven-carbon sugar. Finally, this seven-carbon fragment combines with another three-carbon
fragment to form two additional five-carbon sugars.
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.11. Regeneration of Ribulose 1,5-Bisphosphate. Both ribose 5-phosphate and xylulose 5-phosphate are
converted into ribulose 5-phosphate, which is then phosphorylated to complete the regeneration of ribulose 1,5bisphosphate.
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water
Figure 20.12. Calvin Cycle. The diagram shows the reactions necessary with the correct stoichiometry to convert three
molecules of CO2 into one molecule of DHAP. The cycle is not as simple as presented in Figure 20.1; rather, it entails
many reactions that lead ultimately to the synthesis of glucose and the regeneration of ribulose 1,5-bisphosphate. [After
J. R. Bowyer and R. C. Leegood. "Photosynthesis," in Plant Biochemistry, P. M. Dey and J. B. Harborne, Eds.
(Academic Press, 1997), p. 85.]
II. Transducing and Storing Energy
20. The Calvin Cycle and the Pentose Phosphate Pathway
20.1. The Calvin Cycle Synthesizes Hexoses from Carbon Dioxide and Water