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Biochemistry | © Simon Sindayigaya; contributors: Franco Longhini |
This chapter focus on carbohydrate anabolism, which use chemical energy in the form of ATP and NADH or NADPH to synthesize cellular components from simple precursor molecules. Anabolic pathways are generally reductive rather than oxidative. Catabolism and anabolism proceed simultaneously in a dynamic steady state, so the energy-yielding degradation of cellular components is counterbalanced by biosynthetic processes, which create and maintain the intricate orderliness of living cells. Plants are especially versatile in handling carbohydrates, for several reasons. First, plants are autotrophs, able to convert inorganic carbon (as CO2) into organic compounds. Second, biosynthesis occurs primarily in plastids, membrane-bounded organelles unique to plants, and the movement of intermediates between cellular compartments is an important aspect of metabolism. Third, plants are not motile: they cannot move to find better supplies of water, sunlight, or nutrients. They must have sufficient metabolic flexibility to allow them to adapt to changing conditions in the place where they are rooted. Finally, plants have thick cell walls made of carbohydrate polymers, which must be assembled outside the plasma membrane and which constitute a significant proportion of the cell’s carbohydrate. The chapter begins with a description of the process by which CO2 is assimilated into trioses and hexoses, then considers photorespiration, an important side reaction during CO2 fixation, and the ways in which certain plants avoid this side reaction. We then look at how the biosynthesis of sucrose (for sugar transport) and starch (for energy storage) is accomplished by mechanisms analogous to those employed by animal cells to make glycogen. The next topic is the synthesis of the cellulose of plant cell walls and the peptidoglycan of bacterial cell walls, illustrating the problems of energydependent biosynthesis outside the plasma membrane. Finally, we discuss how the various pathways that share pools of common intermediates are segregated within organelles yet integrated with one another.
The synthesis of carbohydrates in animal cells always
employs precursors having at least three carbons, all of
which are less oxidized than the carbon in CO2. Plants
and photosynthetic microorganisms, by contrast, can
synthesize carbohydrates from CO2 and water, reducing
CO2 at the expense of the energy and reducing power
furnished by the ATP and NADPH that are generated
by the light-dependent reactions of photosynthesis (Fig.
18–1). Plants (and other autotrophs) can use CO2 as the
sole source of the carbon atoms required for the biosynthesis
of cellulose and starch, lipids and proteins, and
the many other organic components of plant cells. By
contrast, heterotrophs cannot bring about the net reduction
of CO2 to achieve a net synthesis of glucose.
Green plants contain in their chloroplasts unique
enzymatic machinery that catalyzes the conversion of
CO2 to simple (reduced) organic compounds, a process
called CO2 assimilation. This process has also been
called CO2 fixation or carbon fixation, but we reserve
these terms for the specific reaction in which CO2
is incorporated (fixed) into a three-carbon organic compound,
the triose phosphate 3-phosphoglycerate. This simple
product of photosynthesis is the precursor of more complex
biomolecules, including sugars, polysaccharides, and
the metabolites derived from them, all of which are synthesized
by metabolic pathways similar to those of animal tissues.
Carbon dioxide is assimilated via a cyclic pathway being constantly regenerated. The pathway
was elucidated in the early 1950s by Melvin Calvin,
Andrew Benson, and James A. Bassham, and is often
called the Calvin cycle or, more descriptively, the
photosynthetic carbon reduction cycle.
Carbohydrate metabolism is more complex in plant
cells than in animal cells or in nonphotosynthetic microorganisms.
In addition to the universal pathways of
glycolysis and gluconeogenesis, plants have the unique
reaction sequences for reduction of CO2 to triose phosphates
and the associated reductive pentose phosphate
pathway—all of which must be coordinately regulated
to ensure proper allocation of carbon to energy production
and synthesis of starch and sucrose.
FIGURE 18–1 Assimilation of CO2 into biomass in plants.
The lightdriven synthesis of ATP and NADPH, described in Chapter 17,
provides energy and reducing power for the fixation of CO2 into trioses,
from which all the carbon-containing compounds of the plant cell are
synthesized. The processes shown with red arrows are the focus of this
chapter.
Most of the biosynthetic activities in plants (including CO2 assimilation) occur in plastids, a family of selfreproducing organelles bounded by a double membrane and containing a small genome that encodes some of their proteins. Most proteins destined for plastids are encoded in nuclear genes, which are transcribed and translated like other nuclear genes; then the proteins are imported into plastids. Plastids reproduce by binary fission, replicating their genome (a single circular DNA molecule) and using their own enzymes and ribosomes to synthesize the proteins encoded by that genome. Chloroplasts are the sites of CO2 assimilation. The enzymes for this process are contained in the stroma, the soluble phase bounded by the inner chloroplast membrane. Amyloplasts are colorless plastids (that is, they lack chlorophyll and other pigments found in chloroplasts). They have no internal membranes analogous to the photosynthetic membranes (thylakoids) of chloroplasts, and in plant tissues rich in starch these plastids are packed with starch granules (Fig. 18–2a). Chloroplasts can be converted to proplastids by the loss of their internal membranes and chlorophyll, and proplastids are interconvertible with amyloplasts (Fig. 18–2b). In turn, both amyloplasts and proplastids can develop into chloroplasts. The relative proportions of the plastid types depend on the type of plant tissue and on the intensity of illumination. Cells of green leaves are rich in chloroplasts, whereas amyloplasts dominate in nonphotosynthetic tissues that store starch in large quantities, such as potato tubers. The inner membranes of all types of plastids are impermeable to polar and charged molecules. Traffic across these membranes is mediated by sets of specific transporters.
FIGURE 18–2 (a) Amyloplasts filled with starch (dark granules) are
stained with iodine in this section of Ranunculus root cells. Starch
granules in various tissues range from 1 to 100 μm in diameter.
(b) 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 internal
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 nonphotosynthetic
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 synthesis
of essential amino acids, thiamine, pyridoxal phosphate, flavins,
and vitamins A, C, E, and K.
The first stage in the assimilation of CO2 into biomolecules (Fig. 18–3) 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-phosphoglycerate 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 carbons) are used to regenerate three molecules of ribulose 1,5-bisphosphate (15 carbons), the starting material. 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. All 13 enzymes of the pathway are found in the chloroplast stroma.
FIGURE 18–3 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.
The plants in which 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 (RuBisCO). The carboxylase catalyzes the covalent attachment of CO2 to the five-carbon sugar ribulose 1,5- bisphosphate and then the unstable six-carbon intermediate is cleavage to form two molecules of 3-phosphoglycerate, one of which bears the carbon introduced as CO2 in its carboxyl group. To achieve high rates of CO2 fixation, plants need large amounts of this enzyme. In fact, RuBisCO makes up almost 50% of soluble protein in chloroplasts and is probably 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 Mg2+ ion brings together and orients the reactants at the active site and polarizes the CO2, opening it to nucleophilic attack by the ribulose ketone functional group (Fig. 18–4). The resulting six-carbon intermediate breaks down to yield two molecules of 3-phosphoglycerate.
FIGURE 18–4 CO2 Fixation: 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. (2) CO2, polarized
by the proximity of the Mg2+ ion, undergoes nucleophilic attack by
the enediolate, producing a branched six-carbon sugar. (3) Hydroxylation
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).
The 3-phosphoglycerate
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. The stromal and cytosolic enzymes
are isozymes; both sets of enzymes catalyze the same
reactions, but they are the products of different genes.
The 3-phosphoglycerate
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 dehydrogenase,
producing glyceraldehyde 3-phosphate and Pi.
Triose phosphate isomerase then interconverts glyceraldehyde
3-phosphate and dihydroxyacetone phosphate.
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 converted
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 (Fig. 18-5). The regeneration of ribulose 1,5-bisphosphate is
accomplished in a series of reactions that, together with stages 1 and 2,
constitute the cyclic pathway.
FIGURE 18–5 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. 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.
One molecule of glyceraldehyde 3-phosphate is the net product of the carbon
assimilation pathway. In summary, for every molecule of triose phosphate
produced by photosynthetic CO2 assimilation, six
NADPH and nine ATP are required. NADPH and
ATP are produced in the lightdependent
reactions of photosynthesis in about the
same ratio (2:3) as they are consumed in the Calvin cycle.
Nine ATP molecules are converted to ADP and phosphate
in the generation of a molecule of triose phosphate;
eight of the phosphates are released as Pi and
combined with eight ADP to regenerate ATP. The ninth
phosphate is incorporated into the triose phosphate itself.
To convert the ninth ADP to ATP, a molecule of Pi
must be imported from the cytosol (Fig. 18-6).
In the dark, the production of ATP and NADPH by
photophosphorylation, and the incorporation of CO2
into triose phosphate
cease. Later in this section we describe the regulatory mechanisms that turn on
carbon assimilation in the light and turn it off in the dark.
FIGURE 18–6 Stoichiometry of CO2 assimilation in the Calvin cycle. For every three CO2
molecules fixed, one molecule of triose phosphate (glyceraldehyde 3-phosphate) is produced
and nine ATP and six NADPH are consumed.
The inner chloroplast membrane have a specific antiporter that catalyzes the one-for-one exchange of Pi with a triose phosphate, either dihydroxyacetone phosphate or 3-phosphoglycerate (Fig. 18–7). This antiporter simultaneously moves Pi into the chloroplast, where it is used in photophosphorylation, and moves triose phosphate into the cytosol, where it can be used to synthesize sucrose, the form in which the fixed carbon is transported to distant plant tissues. Sucrose synthesis in the cytosol and starch synthesis in the chloroplast are the major pathways by which the excess triose phosphate from photosynthesis is “harvested.” Sucrose synthesis releases four Pi molecules from the four triose phosphates required for its production. For every molecule of triose phosphate removed from the chloroplast, one Pi is transported into the chloroplast, providing the ninth Pi mentioned above, to be used in regenerating ATP. If this exchange were blocked, triose phosphate synthesis would quickly deplete the available Pi in the chloroplast, slowing ATP synthesis and suppressing assimilation of CO2 into starch.
FIGURE 18–7 The Pi–triose phosphate antiport system of the inner
chloroplast membrane. This transporter facilitates the exchange of
cytosolic Pi for stromal dihydroxyacetone phosphate. The products of
photosynthetic carbon assimilation are thus moved into the cytosol
where they serve as a starting point for sucrose biosynthesis, and Pi
required for photophosphorylation is moved into the stroma.
The reductive assimilation of CO2 requires a lot of ATP
and NADPH, and their stromal concentrations increase
when chloroplasts are illuminated. Four Calvin cycle enzymes are subject to a
special type of regulation by light. Ribulose 5-phosphate kinase, fructose
1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and glyceraldehyde
3-phosphate dehydrogenase are activated by light-driven reduction of disulfide
bonds between two Cys residues critical to their catalytic activities. When
these Cys residues are disulfidebonded (oxidized), the enzymes are inactive;
this is the normal situation in the dark. With illumination, electrons flow from
photosystem I to ferredoxinnwhich passes electrons to a small, soluble, disulfidecontaining
protein called thioredoxin (Fig. 18–8), in
a reaction catalyzed by ferredoxin-thioredoxin reductase.
When thioredoxin is reduced by the action of ferrodoxin it is thus activated and
in this activated state reduces the critical disulfide bonds in each of the
above mentioned 4 light-regulated
enzymes. These reductive cleavage reactions are
accompanied by conformational changes that increase
enzyme activities.
At nightfall, the Cys residues in the four enzymes are reoxidized to their disulfide forms, the
enzymes are thus inactivated. Instead, starch synthesized and stored
during the daytime is degraded to fuel glycolysis at night.
Glucose 6-phosphate dehydrogenase, the first enzyme
in the oxidative pentose phosphate pathway, is
also regulated by this light-driven reduction mechanism,
but in the opposite sense. During the day, when photosynthesis
produces plenty of NADPH, this enzyme is not
needed for NADPH production. Reduction of a critical
disulfide bond by electrons from ferredoxin inactivates
the enzyme.
FIGURE 18–8 Light activation of several enzymes of the Calvin
cycle. The light activation is mediated by thioredoxin, a small,
disulfide-containing protein. In the light, thioredoxin is reduced by
electrons moving from photosystem I through ferredoxin (Fd) (blue
arrows), then thioredoxin reduces critical disulfide bonds in each
of the enzymes sedoheptulose 1,7-bisphosphatase, fructose 1,6-
bisphosphatase, ribulose 5-phosphate kinase, and glyceraldehye
3-phosphate dehydrogenase, activating these enzymes. In the dark,
the OSH groups undergo reoxidation to disulfides, inactivating the
enzymes.
Photosynthesis in vascular plants takes place in chloroplasts. In the CO2-assimilating reactions (the Calvin cycle), ATP and NADPH are used to reduce CO2 to triose phosphates. These reactions occur in three stages: the fixation reaction itself, catalyzed by rubisco; reduction of the resulting 3-phosphoglycerate to glyceraldehyde 3-phosphate; and regeneration of ribulose 1,5-bisphosphate from triose phosphates. Rubisco condenses CO2 with ribulose 1,5-bisphosphate, forming an unstable hexose bisphosphate that splits into two molecules of 3-phosphoglycerate. Rubisco is activated by covalent modification (carbamoylation of Lys201) catalyzed by rubisco activase and is inhibited by a natural transition-state analog, whose concentration rises in the dark and falls during daylight. Stromal isozymes of the glycolytic enzymes catalyze reduction of 3-phosphoglycerate to glyceraldehyde 3-phosphate; each molecule reduced requires one ATP and one NADPH. The cost of fixing three CO2 into one triose phosphate is nine ATP and six NADPH, which are provided by the light-dependent reactions of photosynthesis. An antiporter in the inner chloroplast membrane exchanges Pi in the cytosol for 3-phosphoglycerate or dihydroxyacetone phosphate produced by CO2 assimilation in the stroma. Oxidation of dihydroxyacetone phosphate in the cytosol generates ATP and NADH, thus moving ATP and reducing equivalents from the chloroplast to the cytosol. Four enzymes of the Calvin cycle are activated indirectly by light and are inactive in the dark, so that hexose synthesis does not compete with glycolysis—which is required to provide energy in the dark.
As we have seen, photosynthetic cells produce O2 (by the splitting of H2O)
and use CO2 to produce 3-phosphoglycerate with a net gaseous change
during photosynthesis that can be written as follow:
CO2 + H2O → O2 + (CH2O)
In the dark plants are carrying out mitochondrial respiration by
the oxidation of substrates to CO2 and the
conversion of O2 to H2O. On top of that, there is another process
in plants that, like mitochondrial respiration, consumes
O2 and produces CO2 and, like photosynthesis, is driven
by light. This process is called photorespiration and is a costly
side reaction of photosynthesis. In this section we describe
this process and the strategies plants use to
minimize its metabolic consequences.
Rubisco is not absolutely specific for CO2 as a substrate. Molecular oxygen
(O2) competes with CO2 at the active site, and about once in every three or four
turnovers, rubisco catalyzes the condensation of O2 with ribulose
1,5-bisphosphate to form 3-phosphoglycerate and 2-phosphoglycolate
(Fig. 18–9), a metabolically
useless product. This is the oxygenase activity
referred as: ribulose
1,5-bisphosphate carboxylase/oxygenase. The reaction
with O2 results in no fixation of carbon and appears to
be a net liability to the cell; salvaging the carbons from
2-phosphoglycolate (by the pathway outlined below)
consumes significant amounts of cellular energy and
releases some previously fixed CO2.
Given that the reaction with oxygen is deleterious to the organism, why did
the evolution of rubisco produce an active site unable to discriminate well
between CO2 and O2? Perhaps much of this evolution occurred before the time,
about 2.5 billion years ago, when production of O2 by photosynthetic organisms
started to raise the oxygen content of the atmosphere. Before that time, there
was no selective pressure for rubisco to discriminate between CO2 and O2. The Km
for CO2 is about 9 μM, and that for O2 is about 350 μM. The modern atmosphere
contains about 20% O2 and only 0.04% CO2, so an aqueous solution in equilibrium
with air at room temperature contains about 250 μMM O2 and 11 μMM CO2—
concentrations that allow significant O2 “fixation” by rubisco
and thus a significant waste of energy. The temperature
dependence of the solubilities of O2 and CO2 is such that
at higher temperatures, the ratio of O2 to CO2
in solution increases. In addition, the affinity of rubisco
for CO2 decreases with increasing temperature, exacerbating
its tendency to catalyze the wasteful oxygenase
reaction. And as CO2 is consumed in the assimilation reactions,
the ratio of O2 to CO2 in the air spaces of a leaf
increases, further favoring the oxygenase reaction.
FIGURE 18–9 Oxygenase activity of rubisco. Rubisco can incorporate
O2 rather than CO2 into ribulose 1,5-bisphosphate. The unstable
intermediate thus formed splits into 2-phosphoglycolate (recycled as
described in Fig. 20–21) and 3-phosphoglycerate, which can reenter
the Calvin cycle.
The glycolate pathway converts two molecules of
2-phosphoglycolate to two molecules of glycine. Glycine
in in prt converted into CO2 and NH3 by the glycine decarboxylase
complex, with the concomitant reduction of NAD+ to NADH
and in part converted into serine via hydroxymethyltransferase.
The overall reaction can be represented as follow:
2 Glycine + NAD++ H2O → serine + CO2 + NH3 + NADH
The serine is converted to hydroxypyruvate, to glycerate,
and finally to 3-phosphoglycerate, which is used to
regenerate ribulose 1,5-bisphosphate, completing the
long, expensive cycle (Fig. 18–10).
The combined activity of the rubisco oxygenase
and the glycolate pathway consumes O2 and
produces CO2—hence the name photorespiration.
This pathway is perhaps better called the oxidative
photosynthetic carbon cycle or C2 cycle.
Unlike mitochondrial respiration, “photorespiration”
does not conserve energy and may
actually reduce biomass formation as much as 50%.
This inefficiency has led to evolutionary adaptations
in the carbon-assimilation processes, particularly in
plants that have evolved in warm climates.
FIGURE 18–10 Glycolate pathway. This pathway, which salvages 2-
phosphoglycolate (shaded pink) by its conversion to serine and eventually
3-phosphoglycerate, involves three cellular compartments. Glycolate
formed by dephosphorylation of 2-phosphoglycolate in
chloroplasts is oxidized to glyoxylate in peroxisomes and then
transaminated to glycine. In mitochondria, two glycine molecules condense
to form serine and the CO2 released during photorespiration
(shaded green). This reaction is catalyzed by glycine decarboxylase,
an enzyme present at very high levels in the mitochondria of C3 plants
(see text). The serine is converted to hydroxypyruvate and then to glycerate
in peroxisomes; glycerate reenters the chloroplasts to be phosphorylated,
rejoining the Calvin cycle. Oxygen (shaded blue) is consumed
at two steps during photorespiration.
In many plants that grow in the tropics (and in temperate climates) a mechanism has evolved to
circumvent the problem of wasteful photorespiration.
The step in which CO2 is fixed into a three-carbon product,
3-phosphoglycerate, is preceded by several steps,
one of which is temporary fixation of CO2 into a four-carbon
compound. Plants that use this process are referred
to as C4 plants, and the assimilation process as
C4 metabolism or the C4 pathway. Plants that use the
carbon-assimilation method we have described so far,
in which the first step is the reaction of CO2 with ribulose
1,5-bisphosphate to form 3-phosphoglycerate, are called
C3 plants.
In tropical
plants, experiments showed that 14CO2 is fixed into oxaloacetate, a four-carbon
compound. This reaction, which occurs in the cytosol of
leaf mesophyll cells, is catalyzed by phosphoenolpyruvate
carboxylase, for which the substrate is HCO3-, not CO2. The oxaloacetate
thus formed is either reduced to malate at the expense of NADPH (as shown in
Fig. 18–11) or converted to aspartate by transamination.
The so formed products are transferred from the mesophyll cells into the
bundle-sheath celles.
In the case of malate the next reaction is further oxidation followed by
decarboxylation to yield pyruvate and CO2 by the action
of malic enzyme, reducing NADP+. In plants that convert,
oxaloacetate to aspartate in the bundle-sheath,
aspartate is reconverted into oxaloacetate and
further reduced to form malate, then
the CO2 is released by malic enzyme or PEP carboxykinase.
Labelling experiments showed that the CO2 released in the bundle-sheath
cells is the same CO2 molecule originally fixed
into oxaloacetate in the mesophyll cells. This CO2 is now
fixed again, this time by rubisco, in exactly the same reaction
that occurs in C3 plants: incorporation of CO2 into
C-1 of 3-phosphoglycerate.
The pyruvate formed by decarboxylation of malate
in bundle-sheath cells is transferred back to the mesophyll
cells, where it is converted to PEP by an unusual
enzymatic reaction catalyzed by pyruvate phosphate
dikinase (Fig. 20–23b). This enzyme is called a dikinase
because two different molecules are simultaneously
phosphorylated by one molecule of ATP: pyruvate to
PEP, and phosphate to pyrophosphate. The pyrophosphate
is subsequently hydrolyzed to phosphate, so
two high-energy phosphate groups of ATP are used in
regenerating PEP. The PEP is now ready to receive another
molecule of CO2 in the mesophyll cell.
The PEP carboxylase of mesophyll cells has a high
affinity for HCO3- (which is favored relative to CO2 in
aqueous solution and can fix CO2 more efficiently than
can rubisco). Unlike rubisco, it does not use O2 as an
alternative substrate, so there is no competition between
CO2 and O2. The PEP carboxylase reaction, then,
serves to fix and concentrate CO2 in the form of malate.
Release of CO2 from malate in the bundle-sheath cells
yields a sufficiently high local concentration of CO2 for
rubisco to function near its maximal rate, and for suppression
of the enzyme’s oxygenase activity.
Once CO2 is fixed into 3-phosphoglycerate in the
bundle-sheath cells, the other reactions of the Calvin cycle
take place exactly as described earlier. Thus in C4
plants, mesophyll cells carry out CO2 assimilation by the
C4 pathway and bundle-sheath cells synthesize starch
and sucrose by the C3 pathway.
The pathway of CO2 assimilation has a greater energy
cost in C4 plants than in C3 plants. For each molecule
of CO2 assimilated in the C4 pathway, a molecule
of PEP must be regenerated at the expense of two highenergy
phosphate groups of ATP. Thus C4 plants need
five ATP molecules to assimilate one molecule of CO2,
whereas C3 plants need only three (nine per triose
phosphate). As the temperature increases (and the
affinity of rubisco for CO2 decreases, as noted above),
a point is reached (at about 28 to 30 C) at which the
gain in efficiency from the elimination of photorespiration
more than compensates for this energetic cost.
FIGURE 18–11 Carbon assimilation in C4 plants.
When rubisco uses O2 rather than CO2 as substrate, the 2-phosphoglycolate so formed is disposed of in an oxygen-dependent pathway. The result is increased consumption of O2—photorespiration or, more accurately, the oxidative photosynthetic carbon cycle or C2 cycle. The 2-phosphoglycolate is converted to glyoxylate, to glycine, and then to serine in a pathway that involves enzymes in the chloroplast stroma, the peroxisome, and the mitochondrion. In C4 plants, the carbon-assimilation pathway minimizes photorespiration: CO2 is first fixed in mesophyll cells into a four-carbon compound, which passes into bundle-sheath cells and releases CO2 in high concentrations. The released CO2 is fixed by rubisco, and the remaining reactions of the Calvin cycle occur as in C3 plants.
During active photosynthesis in bright light, a plant leaf produces more carbohydrate (as triose phosphates) than it needs for generating energy or synthesizing precursors. The excess is converted to sucrose and transported to other parts of the plant, to be used as fuel or stored. In most plants, starch is the main storage form, but in a few plants, such as sugar beet and sugarcane, sucrose is the primary storage form. The synthesis of sucrose and starch occurs in different cellular compartments (cytosol and plastids, respectively), and these processes are coordinated by a variety of regulatory mechanisms that respond to changes in light level and photosynthetic rate.
Starch, like glycogen, is a high molecular weight polymer
of D-glucose in (α1→4) linkage. It is synthesized in
chloroplasts for temporary storage as one of the stable
end products of photosynthesis, and for long-term storage
it is synthesized in the amyloplasts of the nonphotosynthetic
parts of plants—seeds, roots, and tubers
(underground stems).
The mechanism of glucose activation in starch synthesis
is similar to that in glycogen synthesis. An activated
nucleotide sugar, in this case ADP-glucose, is
formed by condensation of glucose 1-phosphate with ATP by
nucleophilic attack of glucose at the α position of ATP displacing
PPi and transferring adenylate (5-AMP) as an adenylyl
group as the activator group. The reaction is made essentially
irreversible by the presence in plastids of inorganic pyrophosphatase.
In summury the reactions are the following:
Glucose 1-phosphate + ATP ⇔ ADP-glucose + PPi
PPi + H20 ⇔ 2 Pi
(the pyrophosphatase reaction is energetically
favourable [-19 kJ/mol] pushing thus the overall reaction to the right)
Starch synthase then transfers glucose residues from
ADP-glucose to preexisting starch molecules. Although it
has generally been assumed that glucose is added to the
nonreducing end of starch, as in glycogen synthesis, evidence now suggests that
starch synthase has two equivalent active sites that alternate in inserting a
glucosyl residue onto the reducing end of the growing chain. This end remains
covalently attached to the enzyme, first at one active site, then at the other
(Fig. 18–12). Attachment to one active site effectively
activates the reducing end of the growing chain for nucleophilic
displacement of the enzyme by the attacking
C-4 hydroxyl of a glucosyl moiety bound to the other active
site, forming the (α1→4) linkage characteristic of
starch.
The amylose of starch is unbranched, but amylopectin
has numerous (α1→6)-linked branches. Chloroplasts contain a branching enzyme,
similar to glycogen-branching enzyme,
that introduces the (α1→6) branches of amylopectin.
Taking into account the hydrolysis by inorganic pyrophosphatase
of the PPi produced during ADP-glucose
synthesis, the overall reaction for starch formation from
glucose 1-phosphate is:
Starchn + glucose 1-phosphate + ATP → starchn + 1 + ADP + 2Pi
ΔG'º = -50 kJ/mol
Many types of bacteria store carbohydrate in the form of glycogen (essentially
highly branched starch), which they synthesize in a reaction analogous to that
catalyzed by glycogen synthase in animals. Bacteria, like plant plastids, use
ADP-glucose as the activated form of glucose, whereas animal cells use
UDP-glucose. Again, the similarity between plastid and bacterial metabolism is
consistent with the endosymbiont hypothesis for the origin of organelles.
FIGURE 18–12 Starch synthesis. Starch synthesis proceeds by a twosite
insertion mechanism, with ADP-glucose as the initial glucosyl
donor. The two identical active sites on starch synthase alternate in
displacing the growing chain from each other, and new glucosyl units
are inserted at the reducing end of the growing chain.
Most of the triose phosphate generated by CO2 fixation in plants is
converted to sucrose or starch.
In the course of evolution, sucrose may have been selected
as the transport form of carbon because of its unusual
linkage between the anomeric C-1 of glucose and
the anomeric C-2 of fructose. This bond is not hydrolyzed
by amylases or other common carbohydrate-cleaving enzymes,
and the unavailability of the anomeric carbons
prevents sucrose from reacting non-enzymatically (as
does glucose) with amino acids and proteins.
Sucrose is synthesized in the cytosol, beginning with the synthesis of fructose
from dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. After
condensation of two triose phosphates to form fructose 1,6- bisphosphate
(catalyzed by aldolase), hydrolysis by fructose
1,6-bisphosphatase yields fructose 6-phosphate. Sucrose 6-phosphate synthase
then catalyzes the reaction of fructose 6-phosphate with UDP-glucose to form
sucrose 6-phosphate (Fig. 18–13). Finally,
sucrose 6-phosphate phosphatase removes the phosphate
group, making sucrose available for export to
other tissues. The reaction catalyzed by sucrose 6-phosphate
synthase is a low-energy process (ΔG'º = -5.7 kJ/mol),
but the hydrolysis of sucrose 6-phosphate to
sucrose is sufficiently exergonic (ΔG'º = -16.5 kJ/mol)
to make the overall synthesis of sucrose essentially
irreversible. One remarkable difference between the cells of
plants and animals is the absence in the plant cell cytosol
of the enzyme inorganic pyrophosphatase, which
catalyzes the reaction:
PPi + H20 ⇔ 2 Pi (ΔG'º = -19.2 kJ/mol)
For many biosynthetic reactions that liberate PPi, pyrophosphatase activity
makes the process more favorable energetically, tending to make these reactions
irreversible.
FIGURE 18–13 Sucrose synthesis. Sucrose is synthesized from
UDPglucose and fructose 6-phosphate, which are synthesized from triose
phosphates in the plant cell cytosol. The sucrose 6-phosphate synthase of most plant
species is allosterically regulated by glucose 6-phosphate and Pi.
Triose phosphates produced by the Calvin cycle in
bright sunlight, as we have noted, may be stored temporarily
in the chloroplast as starch, or converted to sucrose
and exported to nonphotosynthetic parts of the
plant, or both. The balance between the two processes
is tightly regulated, and both must be coordinated with
the rate of carbon fixation. Five-sixths of the triose
phosphate formed in the Calvin cycle must be recycled
to ribulose 1,5-bisphosphate; if more than
one-sixth of the triose phosphate is drawn out of the
cycle to make sucrose and starch, the cycle will slow or
stop. However, insufficient conversion of triose phosphate
to starch or sucrose would tie up phosphate, leaving
a chloroplast deficient in Pi, which is also essential
for operation of the Calvin cycle.
The flow of triose phosphates into sucrose is regulated
by the activity of fructose 1,6-bisphosphatase
(FBPase-1) and the enzyme that effectively reverses its
action, PPi-dependent phosphofructokinase.
These enzymes are therefore critical points for
determining the fate of triose phosphates produced by
photosynthesis. Both enzymes are regulated by fructose
2,6-bisphosphate (F2,6BP), which inhibits FBPase-1
and stimulates PP-PFK-1. In vascular plants, the concentration
of F2,6BP varies inversely with the rate of
photosynthesis (Fig. 20–26). Phosphofructokinase-2,
responsible for F2,6BP synthesis, is inhibited by dihydroxyacetone
phosphate or 3-phosphoglycerate and
stimulated by fructose 6-phosphate and Pi. During active
photosynthesis, dihydroxyacetone phosphate is
produced and Pi is consumed, resulting in inhibition of
PFK-2 and lowered concentrations of F2,6BP. This favors
greater flux of triose phosphate into fructose 6-
phosphate formation and sucrose synthesis. With this
regulatory system, sucrose synthesis occurs when the
level of triose phosphate produced by the Calvin cycle
exceeds that needed to maintain the operation of the
cycle.
The key regulatory enzyme in starch synthesis is
ADP-glucose pyrophosphorylase (Fig. 18–14); it is
activated by 3-phosphoglycerate (which accumulates
during active photosynthesis) and inhibited by Pi (which
accumulates when light-driven condensation of ADP
and Pi slows). When sucrose synthesis slows, 3-phosphoglycerate
formed by CO2 fixation accumulates, activating
this enzyme and stimulating the synthesis of
starch.
FIGURE 18–14 Fructose 2,6-bisphosphate as regulator of sucrose synthesis.
The concentration of the allosteric regulator fructose 2,6-
bisphosphate in plant cells is regulated by the products of photosynthetic
carbon assimilation and by Pi. Dihydroxyacetone phosphate and
3-phosphoglycerate produced by CO2 assimilation inhibit phosphofructokinase-
2 (PFK-2), the enzyme that synthesizes the regulator; Pi
stimulates PFK-2. The concentration of the regulator is therefore
inversely proportional to the rate of photosynthesis. In the dark, the
concentration of fructose 2,6-bisphosphate increases and stimulates
the glycolytic enzyme PPi-dependent phosphofructokinase-1 (PP-PFK-
1), while inhibiting the gluconeogenic enzyme fructose 1,6-
bisphosphatase (FBPase-1). When photosynthesis is active (in the light),
the concentration of the regulator drops and the synthesis of fructose
6-phosphate and sucrose is favored.
Starch synthase in chloroplasts and amyloplasts catalyzes the addition of single glucose residues, donated by ADP-glucose, to the reducing end of a starch molecule by a two-step insertion mechanism. Branches in amylopectin are introduced by a second enzyme. Sucrose is synthesized in the cytosol in two steps from UDP-glucose and fructose 1-phosphate. The partitioning of triose phosphates between sucrose synthesis and starch synthesis is regulated by fructose 2,6-bisphosphate (F2,6BP), an allosteric effector of the enzymes that determine the level of fructose 6-phosphate. F2,6BP concentration varies inversely with the rate of photosynthesis, and F2,6BP inhibits the synthesis of fructose 6-phosphate, the precursor to sucrose.
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