Carbohydrate metabolism in plant cells is more complex than in animal cells or nonphotosynthetic microorganisms. In addition to the universal pathways of glycolysis and gluconeogenesis, plants have the unique reaction sequences for CO2 reduction to triose phosphates and the associated reductive pentose phosphate pathway-all of which must be coordinately regulated to avoid wasteful futile cycling and to ensure proper allocation of carbon to energy production and synthesis of starch and sucrose. One level of coordination is achieved by light activation of certain enzymes associated with the carbon-fixing reactions of photosynthesis. Some enzymes are activated by changes in pH that result from light-induced proton movements; some enzymes are activated by reduction of disulfide bonds involved in their catalytic activity, using electrons flowing from photosystem I; other enzymes are subject to more conventional allosteric regulation by one or more metabolic intermediates. The compartmentation of metabolic sequences in organelles also contributes to the regulation of carbohydrate metabolism.
We will end with a discussion of another reaction catalyzed by rubisco, the condensation of O2 with ribulose-1,5-bisphosphate. This is the starting point for a process called photorespiration, which greatly affects the efficiency of carbon fixation in plants.
Rubisco is also inhibited by 2-carboxyarabinitol-1-phosphate, a naturally occurring transition-state analog (see Box 8-3) with a structure similar to that of the β-keto acid intermediate of the rubisco reaction (Figs. 19-20, 19-30c). This compound is synthesized in the dark by some plants to depress rubisco activity, and it is sometimes called the "nocturnal inhibitor." It is broken down when light returns, permitting a reactivation of rubisco.
Thus, when photosynthesis ceases, the concentration of fructose2,6-bisphosphate rises, stimulating glycolysis and inhibiting gluconeogenesis; glycolysis (combined with mitochondrial oxidative phosphorylation) provides energy in the dark. In the light, the fructose-2,6bisphosphate concentration drops, glycolysis is inhibited, gluconeogenesis is turned on, and the energy and precursors produced by photosynthesis are used to make hexoses to be transported as sucrose or stored as starch.
The salvage pathway (Fig. 19-36) involves the conversion of two molecules of phosphoglycolate into a molecule of serine (which has three carbons) and a molecule of C02. The oxygenase activity of rubisco combined with the salvage pathway consumes 02 and produces CO2, a process called photorespiration. Unlike mitochondrial respiration, however, this process does not conserve energy
C4 plants, which typically grow in high light intensity and temperatures, have several important characteristics: high photosynthetic rates, high growth rates, low photorespiration rates, low rates of water loss, and an unusual leaf structure. Photosynthesis in the leaves of C4 plants involves two cell types: mesophyll and bundle-sheath cells (Fig. 19-37). There are three known patterns of C4 metabolism. The best-understood pathway, worked out in the 1960s by two plant biochemists, Marshall Hatch and Rodger Slack, is described here.
In plants of tropical origin the first intermediate in which 14CO2 is fixed is not 3-phosphoglycerate but oxaloacetate, a four-carbon compound. This reaction, which occurs in leaf mesophyll cells (Fig. 19-37), is catalyzed by phosphoenolpyruvate carboxylase:
In plants that employ aspartate as the carrier of CO2, aspartate is first transaminated to form oxaloacetate then reduced to malate in bundlesheath cells before the release of CO22 by malic enzyme. The free CO2 formed in the bundle-sheath cells is the same CO2 molecule that was originally fixed into oxaloacetate in the mesophyll cells.
In the bundle-sheath cells the CO2arising from the decarboxylation of malate is fixed again (Fig. 19-37), this time by rubisco, in exactly the same reaction that occurs in C3 plants, leading to incorporation of CO2 into the C-1 of 3-phosphoglycerate. The pyruvate formed by the decarboxylation of malate in the bundle-sheath cells is transferred back to the mesophyll cells, where it is converted into PEP by an unusual enzymatic reaction, catalyzed by the enzyme pyruvate phosphate dikinase:
The PEP carboxylase of mesophyll cells has a high affinity for HCO3-- and can fix CO2 more efficiently than can rubisco. Unlike rubisco, PEP carboxylase does not use O2 as an alternative substrate, so there is no competition between CO2 and O2 with this enzyme. This reaction 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, with relatively little of the competitive side reaction with O2.
Once CO2 is fixed into 3-phosphoglycerate in the bundle-sheath cells, all the other reactions of the C3 or Calvin cycle take place exactly as described earlier (Figs. 19-20, 19-22, 19-23). Thus in C4 plants the mesophyll cells carry out CO2 fixation by the C4 pathway, but starch and sucrose biosynthesis occur by the C3 pathway in the bundle-sheath cells.
The pathway of CO2 fixation in C4 plants has a greater energy cost than in C3 plants. For each molecule of CO2 fixed 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 a total of five ATPs to fix one molecule of CO2, whereas C3 plants need only three. As the temperature increases (and the affinity of rubisco for CO2 decreases, as noted above), a point is reached at about 28° to 30°? where the gain in efficiency from the elimination of photorespiration in C4 plants more than compensates for this energetic cost. C4 plants (crabgrass, for example) outgrow most C3 plants during the summer, as any experienced gardener can attest!
We will end with a discussion of another reaction catalyzed by rubisco, the condensation of O2 with ribulose-1,5-bisphosphate. This is the starting point for a process called photorespiration, which greatly affects the efficiency of carbon fixation in plants.
Rubisco Is Subject to Both Positive and Negative Regulation
As the site where photosynthetic CO2 fixation is initiated, rubisco is a prime target for regulation. One type of regulation involves the carbamylation of a Lys residue (Fig. 19-30a, b). At high CO2 levels this occurs nonenzymatically. However, the substrate for this enzyme, ribulose-1,5-bisphosphate, inhibits carbamylation, and this effect is almost complete at physiological CO2 concentrations. An enzyme called rubisco activase overcomes this inhibition and promotes an ATPdependent activation of rubisco that results in carbamylation. The mechanism of activation is unknown, and it is not clear why ATP is required.Rubisco is also inhibited by 2-carboxyarabinitol-1-phosphate, a naturally occurring transition-state analog (see Box 8-3) with a structure similar to that of the β-keto acid intermediate of the rubisco reaction (Figs. 19-20, 19-30c). This compound is synthesized in the dark by some plants to depress rubisco activity, and it is sometimes called the "nocturnal inhibitor." It is broken down when light returns, permitting a reactivation of rubisco.
Certain Enzymes of the Calvin Cycle Are Indirectly Activated by Light
The reductive fixation of CO2 requires ATP and NADPH, and their stromal concentrations increase when chloroplasts are illuminated (Fig. 19-31). The light-induced transport of protons across the thylakoid membrane (Chapter 18) also makes the stromal compartment alkaline and is accompanied by a flow of Mg2+ out of the thylakoid compartment into the stroma. Several stromal enzymes have evolved to take advantage of these light-dependent conditions that signal the availability of ATP and NADPH; they have pH or Mg2+ optima that are better suited to alkaline conditions and high [Mg2+]. Activation of rubisco by formation of the lysyl carbamate is faster at alkaline pH, and high stromal [Mg2+] favors formation of the active Mg2+ complex. Fructose-1,6-bisphosphatase requires Mg2+ and is very dependent upon pH (Fig. 19-32). Its activity increases by a factor of more than 100 when the pH and [Mg2+ ]rise during chloroplast illumination.
Three other enzymes essential to the Calvin cycle's operation are subject to another type of regulation by light. Ribulose-5-phosphate kinase, fructose-1,6-bisphosphatase, and sedoheptulose-1,7-bisphosphatase can exist in either of two forms, differing in the oxidation state of Cys residues essential to their catalytic activity. When these Cys residues are oxidized as disulfide bonds, the enzymes are inactive; this is the normal situation in the dark. With illumination, electrons flow from photosystem I to ferredoxin (see Fig. 18-44), which passes electrons to a small, soluble, disulfide-containing protein called thioredoxin (Fig. 19-33). Thioredoxin donates electrons for the reduction of the disulfide bridges of these light-activated enzymes and is then reactivated in a disulfide-exchange reaction catalyzed by thioredoxin reductase.
Gluconeogenesis and Glycolysis Are Reciprocally Regulated in Plants
The possibility of futile cycling by the simultaneous operation of glycolysis and gluconeogenesis exists in plants as in animals. Plant cells (the chloroplast stroma and cytosol) have all the enzymes of glycolysis, and during dark periods glycolytic breakdown of starch is a major source of energy. During periods of illumination, photosynthetic plant cells produce the triose phosphate intermediates common to glycolysis and gluconeogenesis, and convert these into hexoses, sucrose, and starch as we have just seen. Futile cycling through these two paths is prevented in plants by the regulation of key enzymes of each cytosolic pathway by fructose-2,6-bisphosphate, whose concentration reflects the level of photosynthetic activity.
The concentration of fructose-2,6-bisphosphate varies inversely with the rate of photosynthesis in higher plants (Fig. 19-34). The enzyme phosphofructokinase-2, responsible for fructose-2,6-bisphosphate synthesis, is inhibited by dihydroxyacetone phosphate or 3-phosphoglycerate and stimulated by Pi. During active photosynthesis, dihydroxyacetone phosphate is produced and Pi is consumed, resulting in inhibition of PFK-2 and lowered concentrations of fructose2,6-bisphosphate.
As in animals (Figs. 19-7, 19-8), fructose-2,6-bisphosphate in plants is an activator of glycolysis and an inhibitor of gluconeogenesis (Fig. 19-34). It slows gluconeogenesis by inhibiting the cytosolic fructose-1,6-bisphosphatase, which catalyzes a rate-limiting step in the synthesis of fructose-6-phosphate. It stimulates glycolysis by activating the PP; dependent form of phosphofructokinase-1 (p. 435); this enzyme, a critical control point in glycolysis, is virtually inactive in the absence of fructose-2,6-bisphosphate.Thus, when photosynthesis ceases, the concentration of fructose2,6-bisphosphate rises, stimulating glycolysis and inhibiting gluconeogenesis; glycolysis (combined with mitochondrial oxidative phosphorylation) provides energy in the dark. In the light, the fructose-2,6bisphosphate concentration drops, glycolysis is inhibited, gluconeogenesis is turned on, and the energy and precursors produced by photosynthesis are used to make hexoses to be transported as sucrose or stored as starch.
Sucrose and Starch Synthesis Are Coordinately Regulated
In most plant cells, triose phosphates produced during CO2 fixation are converted primarily to sucrose and starch. The balance between these processes is tightly regulated, and both processes must be coordinated with the rate of carbon fixation. Communication and coordination between sucrose synthesis in the cytosol and carbon fixation and starch synthesis in the chloroplast are mediated by the Pi-triose phosphate antiport system. If sucrose synthesis is too rapid, the transport of the excess Pi into the chloroplast will result in the removal of too much triose phosphate; this will have a deleterious effect on the rate of carbon fixation because five of every six triose phosphate molecules produced in the Calvin cycle are needed to regenerate ribulose-1,5bisphosphate and complete the cycle. If sucrose synthesis is too slow, insufficient Pi will be made available to the chloroplast for triose phosphate synthesis.| Sucrose synthesis is regulated primarily at three steps, those catalyzed by fructose-1,6-bisphosphatase, sucrose-6-phosphate synthase, and sucrose phosphate phosphatase. When light first strikes a leaf in the morning, triose phosphate levels in the cytosol (derived from carbon fixation in the chloroplast) rise. The triose phosphates inhibit the activity of phosphofructokinase-2, and the levels of fructose-2,6bisphosphate fall. This releases the inhibition of fructose-1,6bisphosphatase, permitting the synthesis of fructose-6-phosphate and thus other hexose phosphates, including glucose-6-phosphate. Glucose-6-phosphate is an allosteric activator of sucrose-6-phosphate synthase. Sucrose phosphate phosphatase catalyzes the final step in sucrose synthesis as its substrate becomes available. The key regulatory enzyme in starch synthesis is ADP-glucose pyrophosphorylase (Fig. 19-16), which is indirectly activated as cytosolic sucrose levels rise. Sucrose-6-phosphate synthase and sucrose phosphate phosphatase are both inhibited to some degree by sucrose. This inhibition becomes effective as sucrose levels increase, leading to an increase in the cytosolic pool of hexose phosphates that effectively sequesters much of the available phosphate in a form that cannot be transported back to the chloroplast. The levels of free Pi in the cytosol, and the resulting decrease in the activity of the Pi-triose phosphate antiporter, lead to a decrease in Pi and an increase in three-carbon compounds in the chloroplast. ADP-glucose pyrophosphorylase is inhibited by Pi and stimulated by 3-phosphoglycerate, so that one effect of the increase in cytosolic sucrose concentration is an increase in starch synthesis in the chloroplast. In the steady state, all of these activities are balanced so that sucrose and starch synthesis each consume about 50?0 of the triose phosphate produced during carbon fixation, and the rates of both processes are regulated so that precursors and intermediates required for carbon fixation are maintained at optimal levels. |
Condensation of O2 with Ribulose-1,5-Bisphosphate Initiates Photorespiration
Rubisco is not absolutely specific for CO2 as a substrate; O2 competes with CO2 at the active site, and rubisco catalyzes the condensation of O 2 with ribulose-1,5-bisphosphate to form one molecule of 3-phosphoglycerate and one of phosphoglycolate (Fig. 19-35). This is the enzyme's oxygenase activity, evident in its name: RuBP carboxylase/oxygenase. The metabolic function of this reaction is not clear. It results in no fixation of carbon and appears to be a net liability to the cell in which it occurs; salvaging the carbons from phosphoglycolate, which is not a useful metabolite, uses cellular energy. The condensation of 02 1with ribulose-1,5-bisphosphate occurs concurrently with C02 fixation, with the latter predominating by a factor of about three.The salvage pathway (Fig. 19-36) involves the conversion of two molecules of phosphoglycolate into a molecule of serine (which has three carbons) and a molecule of C02. The oxygenase activity of rubisco combined with the salvage pathway consumes 02 and produces CO2, a process called photorespiration. Unlike mitochondrial respiration, however, this process does not conserve energy
| Apparently the evolution of rubisco produced an active site not able to discriminate well between CO2 and O2, perhaps because much evolution occurred before O2 was an important component of the atmosphere. The Km for CO2 is about 20μM, and for O2 is about 200 μM. (A solution that is in equilibrium with air at room temperature contains about 10 μM CO2 and 250 μM O2.) The modern atmosphere contains about 20% O2 and only 0.04% CO2, proportions that allow O2 "fixation" by rubisco to constitute a significant waste of energy. During photosynthesis CO2 is consumed in the fixation reactions, altering the CO2 to O2 ratio in the air spaces around the leaf in favor of O2. In addition, the affinity of rubisco for CO2 decreases with increasing temperature, exacerbating the tendency of the enzyme to catalyze the wasteful oxygenase reaction. Photorespiration may inhibit net biomass formation as much as 50%. As we shall see, this has led to adaptations in the process by which carbon fixation takes place, particularly in plants that live in warm climates. Some Plants Have a Mechanism to Prevent Photorespiration Most plants in the tropics, as well as temperate-zone crop plants native to the tropics, such as corn, sugar cane, and sorghum, have evolved a mechanism to circumvent the problem of wasteful photorespiration. The ultimate step of CO2 fixation into a three-carbon product, 3-phosphoglycerate, is preceded by several steps, one of which is a preliminary fixation of CO2 into a compound with four carbon atoms. These plants are referred to as C4 plants. Plants in which the first step in carbon fixation is reaction of CO2 with ribulose-1,5-bisphosphate to form 3-phosphoglycerate-as we have described thus far-are called C3 plants. |
In plants of tropical origin the first intermediate in which 14CO2 is fixed is not 3-phosphoglycerate but oxaloacetate, a four-carbon compound. This reaction, which occurs in leaf mesophyll cells (Fig. 19-37), is catalyzed by phosphoenolpyruvate carboxylase:
Phosphoenolpyruvate + HCO3-- ==== oxaloacetate + Pi
This enzyme does not occur in animal tissues and is not to be confused with phosphoenolpyruvate carboxykinase (p. 603), which catalyzes the gluconeogenic reaction
Oxaloacetate + GTP ===== phosphoenolpyruvate + CO2 + GDP
The oxaloacetate formed in the mesophyll cells is reduced to malate at the expense of NADPH:
Oxaloacetate + NADPH + H+; L-malate +NADP+
| Alternatively, oxaloacetate may be converted to aspartate by transamination:
Oxaloacetate + α-amino acid ===== L-aspartate + α-keto acid The malate or aspartate formed in the mesophyll cells, which contains the fixed CO2, is transferred into the neighboring bundle-sheath cells via specialjunctions (plasmodesmata; p. 49) between the cells (Fig. 1937). In the bundle-sheath cells the malate is oxidized and decarboxylated to yield pyruvate and CO2 by the action of malic enzyme:
L-Malate + NADP+ ===== pyruvate + CO2 + NADPH + H+
|
In the bundle-sheath cells the CO2arising from the decarboxylation of malate is fixed again (Fig. 19-37), this time by rubisco, in exactly the same reaction that occurs in C3 plants, leading to incorporation of CO2 into the C-1 of 3-phosphoglycerate. The pyruvate formed by the decarboxylation of malate in the bundle-sheath cells is transferred back to the mesophyll cells, where it is converted into PEP by an unusual enzymatic reaction, catalyzed by the enzyme pyruvate phosphate dikinase:
Pyruvate + ATP + Pi======= phosphoenolpyruvate + AMP + PPi
This enzyme is called a dikinase because two different molecules are simultaneously phosphorylated by one molecule of ATP: pyruvate is phosphorylated to PEP, and phosphate is phosphorylated 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 fix another molecule of CO2 in the mesophyll cell.The PEP carboxylase of mesophyll cells has a high affinity for HCO3-- and can fix CO2 more efficiently than can rubisco. Unlike rubisco, PEP carboxylase does not use O2 as an alternative substrate, so there is no competition between CO2 and O2 with this enzyme. This reaction 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, with relatively little of the competitive side reaction with O2.
Once CO2 is fixed into 3-phosphoglycerate in the bundle-sheath cells, all the other reactions of the C3 or Calvin cycle take place exactly as described earlier (Figs. 19-20, 19-22, 19-23). Thus in C4 plants the mesophyll cells carry out CO2 fixation by the C4 pathway, but starch and sucrose biosynthesis occur by the C3 pathway in the bundle-sheath cells.
The pathway of CO2 fixation in C4 plants has a greater energy cost than in C3 plants. For each molecule of CO2 fixed 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 a total of five ATPs to fix one molecule of CO2, whereas C3 plants need only three. As the temperature increases (and the affinity of rubisco for CO2 decreases, as noted above), a point is reached at about 28° to 30°? where the gain in efficiency from the elimination of photorespiration in C4 plants more than compensates for this energetic cost. C4 plants (crabgrass, for example) outgrow most C3 plants during the summer, as any experienced gardener can attest!
