LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL. 2. BIOENERGETICS AND METABOLISM - 2014

PART II. BIOENERGETICS AND METABOLISM

Class="center">...the discovery by Samuel Ruben and Martin Kamen in 1940 of the long-lived carbon isotope ¹⁴C provided the ideal tool for tracing the pathway taken by carbon dioxide on its way to carbohydrate.

Melvin Calvin, Nobel Lecture, 1961

20. BIOSYNTHESIS OF CARBOHYDRATES IN PLANTS AND BACTERIA

We have now reached a turning point in our Study of Cellular METABOLISM. Up to this point in Part II, we have described how CARBOHYDRATES, Fatty acids, and Amino Acids—the major metabolic fuels—are broken down via converging Catabolic pathways to enter The Citric Acid Cycle and deliver their electrons to the Respiratory Chain; and how this exothermic flow of electrons to oxygen is coupled to the endothermic synthesis of ATP. We now turn to anabolic pathways, which utilize 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 anabolic processes occur simultaneously, so that in a dynamically

steady state, The breakdown of energy-yielding cellular compounds is balanced by biosynthetic processes that create and maintain the Organization of living Cells.

Plants must be among the most versatile living systems in terms of Carbohydrate Metabolism for several reasons. First, as autotrophs, plants are capable of converting inorganic carbon (such as CO₂) into Organic compounds. Second, Biosynthesis occurs largely in Plastids—membrane-bound Organelles unique to plants—and The transport of intermediates between cellular compartments is a crucial aspect of their metabolism. Third, plants are sessile: they cannot move in search of more accessible Water, sunlight, or nitrates. They must possess sufficient metabolic lability to adapt to the changing conditions of their growing environment. Finally, the multilayered plant Cell wall consists of high-molecular-weight carbohydrates (natural Biopolymers) that must be assembled on the outside of The Plasma Membrane, accounting for a significant fraction of all cellular carbohydrates.

We begin the chapter by describing the processes through which CO₂ is converted into trioses and hexoses, followed by Photorespiration—an important side reaction in CO₂ fixation—and the pathways by which certain plants bypass this side reaction. We then examine how The biosynthesis of sucrose (for sugar transport) and starch (for energy storage) proceeds via mechanisms analogous to those animals use in Glycogen synthesis. The next topic discussed, the synthesis of plant cell wall Cellulose and Introduction/37.html">Bacterial cell wall peptidoglycan, illustrates the challenges of energy-dependent biosynthesis occurring on the exterior of the plasma membrane. Finally, we discuss how various pathways utilizing pools of common intermediates, segregated within organelles, ultimately become integrated with one another.

20.1. Photosynthetic Carbohydrate Synthesis

In animal cells, the synthesis of carbohydrates always relies on precursors containing at least three carbon atoms, which are less oxidized than the carbon in CO₂. In contrast, plants and photosynthetic microorganisms can synthesize carbohydrates from CO₂ and water by reducing CO₂ at the expense of ATP and NADPH generated during the light-dependent reactions of Photosynthesis (Fig. 20-1). Plants (and other autotrophs) can use CO₂ as the sole source of carbon atoms required for the biosynthesis of cellulose, starch, Lipids, Proteins, and many other organic Components of the plant cell. Heterotrophs, by contrast, are incapable of carrying out CO₂ reduction reactions to synthesize glucose.

Fig. 20-1. Incorporation of CO₂ into plant biomass. The light-dependent synthesis of NADPH and ATP, described in Chapter 19, provides the energy and reducing power for the fixation of CO₂ into trioses, from which all carbon-containing compounds of The plant cell are synthesized. This process (indicated by the red arrows) is discussed in detail in this chapter.

Green plants contain a unique enzyme complex within their Chloroplasts that catalyzes The conversion of СO₂ into simple (reduced) organic compounds. This process is referred to as СO₂ assimilation, СO₂ fixation, or carbon fixation. We will use these terms to describe the specific reaction in which СO₂ is incorporated (fixed) into a 3-carbon compound, triose phosphate-3-phosphoglycerate. This simple product of photosynthesis serves as a precursor to more complex Biomolecules (sugars, Polysaccharides, and metabolites) synthesized via metabolic pathways similar to those found in animal Tissues. Carbon dioxide СO₂ is assimilated through a cyclic pathway in which its Key Intermediates are continuously regenerated. This pathway was elucidated in the early 1950s by Melvin Calvin, Andrew Benson, and James A. Bassham, and is commonly known as the Calvin cycle or, more descriptively, the photosynthetic carbon reduction cycle.

Melvin Calvin, 1911–1997

Carbohydrate Metabolism in plant cells is considerably more complex than in animal cells or nonphotosynthetic microorganisms. In addition to the standard pathways of Glycolysis and Gluconeogenesis, plants operate a unique sequence of reactions to reduce СO₂ to triose phosphates, alongside the associated reductive Pentose Phosphate Pathway. All of these reactions must be coordinately regulated to ensure the proper partitioning of carbon atoms between energy-producing reactions and the synthesis of starch and sucrose. As we will see, Key Enzymes are regulated by: (1) the reduction of Disulfide Bonds via electrons originating from Photosystem I, and (2) light-induced changes in pH and magnesium ion concentration. Examining Other Aspects of plant carbohydrate metabolism, we also find enzymes controlled by (3) conventional Allosteric Regulation involving one or more metabolic intermediates, and (4) covalent modification (phosphorylation).

Plastids Are Unique Organelles of PLANT CELLS AND Algae

Most plant biosynthetic processes (including СO₂ assimilation) take place in plastids—a family of self-replicating organelles bounded by a double membrane and containing a small genome that encodes a subset of plastid proteins. Proteins destined for plastids are typically encoded by nuclear genes, which are transcribed and translated like other nuclear genes, and subsequently imported into the plastids. Plastids reproduce by binary fission, replicating their genome (a single circular DNA molecule) and utilizing their own enzymes and Ribosomes to synthesize proteins encoded by that genome. Chloroplasts (see Fig. 19-45) serve as the centers of СO₂ assimilation. The proteins required for this process are located in the stroma, the soluble phase enclosed by the inner chloroplast membrane. Amyloplasts are colorless plastids lacking chlorophyll and other pigments found in chloroplasts. They lack the internal membrane analogs of the photosynthetic membranes (thylakoids) characteristic of chloroplasts; in starch-rich plant tissues, these plastids are packed with starch granules (Fig. 20-2). Chloroplasts can lose their internal membranes and chlorophyll to become proplastids, while proplastids can develop into amyloplasts (Fig. 20-3). Conversely, both amyloplasts and proplastids can differentiate back into chloroplasts. The relative Abundance of different plastid types depends on the plant tissue type and light intensity. Green leaf cells are rich in chloroplasts, whereas nonphotosynthetic tissues, such as potato tubers, are dominated by amyloplasts laden with abundant starch reserves.

Fig. 20-2. Iodine-stained starch granules (dark deposits) within amyloplasts of buttercup (Ranunculus) ROOT cells. The diameter of starch granules varies from 1 to 100 µm across different cell types.

Fig. 20-3. Plastids: origin and interconversion. Plastids of all types are bounded by a double membrane, and some (particularly mature chloroplasts) possess additional internal membranes. These internal membranes can be degraded (when a mature chloroplast converts into a proplastid) and synthesized anew (as when a proplastid develops into a pregranal plastid and subsequently a mature chloroplast). Proplastids in nonphotosynthetic tissues (such as roots) differentiate into amyloplasts, which accumulate large amounts of starch. All plants contain plastids; these organelles host vital processes such as the synthesis of amino acids, thiamine, Pyridoxal phosphate, flavins, and Vitamins A, C, E, and K.

The inner membrane of all plastid types is impermeable to polar and charged molecules. Membrane transport is mediated by a suite of specific transporters.

Carbon Dioxide Assimilation Occurs in Three Stages

The First stage of CO₂ assimilation into biomolecules (Fig. 20-4) is the carbon fixation reaction: the Condensation of CO₂ with the five-carbon acceptor ribulose 1,5-bisphosphate to yield two molecules of 3-phosphoglycerate. In the second stage, 3-phosphoglycerate is reduced to triose phosphates. Overall, three molecules of CO₂ are fixed into three molecules of ribulose 1,5-bisphosphate, producing six molecules of glyceraldehyde 3-phosphate (18 carbon atoms) in equilibrium with dihydroxyacetone phosphate. In the Third Stage, five of the six triose phosphate molecules (15 carbon atoms) are used to regenerate the three molecules of ribulose 1,5-bisphosphate (15 carbon atoms), the starting compound. The sixth molecule of triose phosphate, the end product of photosynthesis, can be used to synthesize hexoses—serving as fuel and structural Materials—sucrose for transport to nonphotosynthetic tissues, or starch for storage. Thus, the entire process is cyclic, with the continuous conversion of CO₂ into triose or hexose phosphates. Fructose 6-phosphate is a key intermediate in the third stage of CO₂ assimilation; it sits at a branch point leading either to the regeneration of ribulose 1,5-bisphosphate or to starch synthesis. The pathway from hexose phosphate to pentose bisphosphate involves many of the same reactions used in animal cells to convert pentose phosphates into hexose phosphates in the nonoxidative phase of the pentose phosphate pathway (see Fig. 14-22). During photosynthetic CO₂ assimilation, many of these reactions operate in reverse, converting hexose phosphates into pentose phosphates. This reductive pentose phosphate pathway utilizes the same enzymes as the oxidative pathway, along with a few additional enzymes that render the reductive cycle irreversible. All 13 enzymes of this pathway are located in the chloroplast stroma.

Fig. 20-4. The three stages of CO₂ assimilation in photosynthetic organisms. The stoichiometry (numbers in parentheses) of three key intermediates determines The Fate of the carbon atoms entering and leaving the cycle: three molecules of CO₂ are incorporated into the carbon Skeleton of one molecule of glyceraldehyde 3-phosphate. This cycle is known as the photosynthetic carbon reduction cycle, or The Calvin Cycle.

Stage 1. Fixation of CO₂ onto 3-phosphoglycerate.

The core secret to the mechanisms of CO₂ assimilation in photosynthetic organisms was unraveled in the late 1940s. Calvin and his coworkers exposed a suspension of green algae to radiolabeled carbon dioxide (¹⁴CO₂) for just a few seconds, then rapidly quenched the cells, extracted their contents, and used chromatographic Methods to identify the metabolites in which the radiolabeled carbon first appeared. The initial compound found to contain the radioactive label was 3-phosphoglycerate, predominantly with the ¹⁴C label at the carboxyl group. These experiments provided compelling evidence that 3-phosphoglycerate is one of the primary intermediates in photosynthesis. Plants in which this three-carbon compound is the first intermediate are called C₃ plants, to distinguish them from the C₄ plants described below.

The incorporation of CO₂ into organic compounds is catalyzed by the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase, commonly abbreviated as rubisco. As a carboxylase, rubisco catalyzes the covalent attachment of CO₂ to the five-carbon sugar ribulose 1,5-bisphosphate and cleaves the resulting unstable six-carbon intermediate to yield two molecules of 3-phosphoglycerate, one of which bears the carbon introduced as CO₂ in its carboxyl group (Fig. 20-4). The oxygenase activity of the enzyme is discussed in Section 20.2.

There are two distinct forms of rubisco. Form I is found in vascular plants, algae, and cyanobacteria, whereas Form II is restricted to photosynthetic Bacteria. Plant rubisco, the key enzyme responsible for biomass formation from CO₂, is Form I (Fig. 20-5a)—consisting of eight identical large subunits (M_r = 53,000; encoded in the Chloroplast Genome, or plastome; each subunit containing a catalytic site) and eight identical small subunits (M_r = 14,000; encoded in the nuclear genome) of undetermined function. Form II rubisco from photosynthetic bacteria has a simpler architecture, containing two subunits that closely resemble the large subunits of the plant enzyme (Fig. 20-5b). This similarity is consistent with the Endosymbiotic Hypothesis for THE ORIGIN OF chloroplasts (p. 59, vol. 1). A notable feature of the plant enzyme is its low turnover number: rubisco fixes only about three molecules of CO₂ per second at 25 °C. To achieve a high rate of CO₂ fixation, plants must maintain large amounts of this enzyme. Rubisco accounts for nearly 50% of the soluble protein in chloroplasts and is arguably one of the most abundant proteins in the biosphere.

Fig. 20-5. Structure of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco). (a) Top and side views of the ribbon model of Form I rubisco from spinach (PDB ID 8RUC). The enzyme comprises eight large subunits (blue) and eight small subunits (gray), tightly packed into a structure with an M_r of 550,000. The concentration of rubisco in the chloroplast stroma is approximately 250 mg/mL, corresponding to an unusually high active-site concentration (~4 mM). Active-site amino acid residues are shown in yellow, and Mg²⁺ ions in green. (b) Ribbon model of Form II rubisco from the bacterium Rhodospirillum rubrum (PDB ID 9RUB). Subunits are shown in gray and blue. The active-site Lys residue, which is carboxylated to a carbamate in the active enzyme, is highlighted in red. The substrate, ribulose 1,5-bisphosphate, is shown in yellow, and magnesium ions in green.

In the proposed mechanism for plant rubisco, a carbamoylated Lys side chain and an associated Mg²⁺ ion play a central role. The Mg²⁺ ion helps properly orient the reactants in the Active Site (Fig. 20-6) and polarizes CO₂, priming it for nucleophilic attack by the five-carbon enediolate active-site intermediate generated on the enzyme (Fig. 20-7). The resulting six-carbon intermediate breaks down to yield two molecules of 3-phosphoglycerate.

Fig. 20-6. The Central Role of the Mg²⁺ ion in the Catalytic Mechanism of rubisco (from PDB ID 1RXO). The Mg²⁺ ion is coordinated by six oxygen atoms occupying the vertices of an octahedral complex: the carbamate oxygen of Lys²⁰¹, two carboxyl oxygen atoms from Glu²⁰⁴ and Asp²⁰³, two oxygens at the C-2 and C-3 positions of the ribulose 1,5-bisphosphate substrate, and one oxygen atom from the second substrate, CO₂. In this crystal structure, a water molecule occupies the CO₂-binding site. (Residue numbers correspond to the spinach enzyme.)

Fig. 20-7. Reaction mechanism. The first stage of CO₂ assimilation: the carboxylase activity of rubisco. The CO₂ fixation reaction is catalyzed by ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco): one molecule of CO₂ combines with one molecule of ribulose 1,5-bisphosphate to yield two molecules of 3-phosphoglycerate, one of which contains the carbon atom from CO₂ (red). Additional proton transfer (not shown) involves Lys²⁰¹, Lys¹⁷⁵, and His²⁹⁴ and proceeds in several steps. Mechanism of rubisco action

As the catalyst for the first stage of photosynthetic CO₂ assimilation, rubisco serves as a primary target for regulation. The enzyme remains inactive until the ε-amino group of Lys²⁰¹ is carbamoylated (Fig. 20-8). Ribulose 1,5-bisphosphate inhibits carbamoylation by binding tightly to the active site and locking the enzyme in a "closed" conformation in which Lys²⁰¹ is inaccessible. Rubisco activase relieves this inhibition via ATP-dependent release of ribulose 1,5-bisphosphate, exposing the Lys amino group for nonenzymatic carbamoylation by CO₂; this process is followed by the binding of Mg²⁺, which activates rubisco. In some species, rubisco activase is light-activated via a redox mechanism (see Fig. 20-19).

Fig. 20-8. The Role of rubisco activase in the carbamoylation of the active-site Lys²⁰¹ residue of rubisco. When the substrate ribulose 1,5-bisphosphate is bound to the active site, Lys²⁰¹ is inaccessible. Rubisco activase hydrolyzes ATP and ejects the sugar bisphosphate, thereby exposing Lys²⁰¹ to the exterior; this Lys residue can now be carbamoylated by a CO₂ molecule (apparently a nonenzymatic reaction). The Mg²⁺ ion is attracted to the negatively charged carbamoyl-Lys and binds to it, leading to enzyme activation.

Another regulatory mechanism involves the "dark inhibitor" 2-carboxyarabinitol 1-phosphate, a naturally occurring transition-state analogue (see Box 6-3, Vol. 1) with a structure resembling the β-keto acid intermediate in the rubisco reaction (Fig. 20-7; see also Fig. 20-20). This compound, synthesized by some plants in the dark, is a potent inhibitor of carbamoylated rubisco. It is either broken down upon illumination or displaced by rubisco activase, thereby activating rubisco.

Stage 2. Conversion of 3-phosphoglycerate to glyceraldehyde 3-phosphate.

The 3-phosphoglycerate formed in the first stage is converted into glyceraldehyde 3-phosphate in two steps, which are essentially the reverse of the corresponding glycolytic reactions with one exception: NADPH is used instead of NADH as the nucleotide cofactor for the reduction of 1,3-bisphosphoglycerate (see Fig. 20-9). The chloroplast stroma contains all glycolytic enzymes except phosphoglycerate mutase. The stromal and cytosolic enzymes are isozymes: both sets of enzymes catalyze identical reactions, but they are encoded by different genes.

Fig. 20-9. The Second Stage of CO₂ assimilation. 3-Phosphoglycerate is converted into glyceraldehyde 3-phosphate (red arrows). Also shown are alternative pathways involving the fixed carbon of glyceraldehyde 3-phosphate (blue arrows). Glyceraldehyde 3-phosphate is predominantly converted back into ribulose 1,5-bisphosphate, as shown in Fig. 20-10. Small amounts of "extra" glyceraldehyde 3-phosphate may be used immediately as an energy source, but all remaining glyceraldehyde 3-phosphate is converted into sucrose for transport or stored as starch within the chloroplasts. In the latter case, it condenses in the stroma with dihydroxyacetone phosphate to form fructose 1,6-bisphosphate, a starch precursor. In other situations, glyceraldehyde 3-phosphate is converted into dihydroxyacetone phosphate, which leaves the chloroplast via a specific transporter (see Fig. 20-15) and can be broken down with energy release via glycolysis in the Cytosol or used to form fructose 6-phosphate and, consequently, sucrose.

In the first reaction of the second stage, the stromal enzyme 3-phosphoglycerate kinase catalyzes The transfer of a phosphoryl group from ATP to 3-phosphoglycerate, converting it into 1,3-bisphosphoglycerate. Subsequently, the chloroplast-specific isozyme glyceraldehyde-3-phosphate dehydrogenase reduces the 1,3-bisphosphate, oxidizing NADPH, to yield glyceraldehyde 3-phosphate and inorganic phosphate P_i. Triose phosphate isomerase catalyzes the interconversion of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Most of the triose phosphate molecules produced in this manner are then used to regenerate ribulose 1,5-

bisphosphate; the remainder are either converted into starch within the chloroplasts and stored for future use, or immediately exported to the cytosol and converted into sucrose for transport to growing plant Organs. In developing leaves, a significant fraction of the triose phosphate may be broken down via glycolysis to release energy.

Stage 3. Regeneration of ribulose 1,5-bisphosphate from triose phosphates.

The first reaction of СO₂ assimilation into triose phosphates consumes ribulose 1,5-bisphosphate, and for the continuous conversion of СO₂ into carbohydrates, ribulose 1,5-bisphosphate must be constantly regenerated. This is accomplished through a series of reactions (Fig. 20-10) which, together with stages (1) and (2), constitute the cyclic pathway shown in Fig. 20-4. The product of the first assimilation reaction (3-phosphoglycerate) undergoes various transformations that ultimately yield ribulose 1,5-bisphosphate. The intermediates of this pathway are three-, four-, five-, six-, and seven-carbon sugars. In the subsequent Discussion, the stage numbers correspond to Fig. 20-10.

Fig. 20-10. Third stage of СO₂ assimilation. The interconversion of triose phosphates and pentose phosphates is shown. Black dots indicate carbon atoms. Glyceraldehyde 3-phosphate and dihydroxyacetone phosphate serve as starting materials. The synthesis reactions of pentose phosphates are catalyzed by transaldolase ((1) and (4)) and transketolase ((3) and (6)). Ribulose 1,5-phosphate is then formed from ribose 5-phosphate by the action of ribose 5-phosphate isomerase ((7)) and from xylulose 5-phosphate by the action of ribulose 5-phosphate epimerase ((8)). At stage (9), ribulose 5-phosphate is phosphorylated to yield ribulose 1,5-bisphosphate. The reactions depicted by blue arrows (stages (2) fructose 1,6-bisphosphatase, (5) sedoheptulose bisphosphatase, and (9) ribulose 5-phosphate kinase) are endothermic, making the overall process irreversible.

Stages (1) and (4) are catalyzed by the same enzyme, aldolase. First, this enzyme catalyzes the reversible condensation of glyceraldehyde 3-phosphate with dihydroxyacetone phosphate to form fructose 1,6-bisphosphate; at stage (2), this compound is cleaved by fructose 1,6-bisphosphatase (FBPase-1) into fructose 6-phosphate and P_i. This reaction is highly exothermic and essentially irreversible. Stage (3) is catalyzed by transketolase, which contains thiamine pyrophosphate (TPP) as a prosthetic group (see Fig. 14-14, a) and requires Mg²⁺ for its function. Transketolase catalyzes the reversible transfer of a two-carbon ketol group (CH₂OH-CO-) from the ketose phosphate donor fructose 6-phosphate to the aldose phosphate acceptor glyceraldehyde 3-phosphate (Fig. 20-11, a, b), yielding the pentose xylulose 5-phosphate and the tetrose erythrose 4-phosphate. At stage (4), transaldolase acts again, condensing erythrose 4-phosphate with dihydroxyacetone phosphate to form the seven-carbon sugar sedoheptulose 1,7-bisphosphate. The enzyme sedoheptulose 1,7-bisphosphatase, which is unique to plastids, converts this bisphosphate into sedoheptulose 7-phosphate (stage (5)); in this pathway, this is the second irreversible reaction. Transketolase then operates again, converting sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate into two pentose phosphates at stage (6) (Fig. 20-11, c). Figure 20-12 shows how a two-carbon fragment is temporarily transferred at stage (6) to the transketolase cofactor TPP and condensed with the three carbons of glyceraldehyde 3-phosphate.

Fig. 20-11. Calvin cycle Reactions Catalyzed by transketolase. (a) The reaction catalyzed by transketolase, involving the transfer of a two-carbon group—temporarily carried by enzyme-bound TPP—from a keto donor to an aldo acceptor. (b) Conversion of a hexose and a triose into a four-carbon and a five-carbon sugar (stage (3) in Fig. 20-10). (c) Conversion of a seven-carbon and a three-carbon sugar into pentoses (stage (6) in Fig. 20-10).

Fig. 20-12. Thiamine pyrophosphate (TPP) acts as a cofactor for transketolase. Transketolase transfers a two-carbon group from sedoheptulose 7-phosphate to glyceraldehyde 3-phosphate, producing two pentose phosphates (stage (6) in Fig. 20-10). Thiamine pyrophosphate facilitates this interaction by temporarily acting as a carrier for the two-carbon structure and stabilizing the electron (see Fig. 14-14).

Fig. 20-13. Regeneration of ribulose 1,5-bisphosphate. Ribulose 1,5-bisphosphate (the Starting Material for the Calvin cycle) is derived from two pentose phosphates generated in the cycle. These reactions involve isomerases and epimerases, followed by phosphorylation by a kinase, with ATP serving as the phosphate group donor (stages (7), (8), and (9) in Fig. 20-10).

The pentose phosphates ribose 5-phosphate and xylulose 5-phosphate, produced in the transketolase reactions, are converted into ribulose 5-phosphate (stages (7) and (8)). In The final stage of the cycle ((9)), this is phosphorylated to ribulose 1,5-bisphosphate by ribulose 5-phosphate kinase (Fig. 20-13). This is the third exothermic reaction of the pathway, as it involves the "replacement" of the high-energy phosphoanhydride bond of ATP with the phosphoester bond in ribulose 1,5-bisphosphate.

The synthesis of each triose phosphate from СO₂ requires six NADPH and nine ATP

Three turns of the Calvin cycle, utilizing three molecules of СO₂ and one molecule of phosphate, yield one molecule of triose phosphate. The Stoichiometry of the complete pathway from СO₂ to triose phosphate with the regeneration of ribulose 1,5-bisphosphate is illustrated in Fig. 20-14. Three molecules of ribulose 1,5-bisphosphate (15 carbon atoms in total) condense with three molecules of СO₂ (three carbon atoms) to form six molecules of 3-phosphoglycerate (18 carbon atoms). These six molecules of 3-phosphoglycerate are reduced to six molecules of glyceraldehyde 3-phosphate (or dihydroxyacetone phosphate), consuming six ATP (during the synthesis of 1,3-bisphosphoglycerate) and six NADPH (during the reduction of 1,3-bisphosphoglycerate to glyceraldehyde 3-phosphate). The glyceraldehyde 3-phosphate dehydrogenase isozyme present in chloroplasts can use NADP⁺ as an electron carrier and normally Functions in the direction of 1,3-bisphosphoglycerate reduction. The cytosolic isozyme uses NAD⁺ as a glycolytic enzyme in animals and other eukaryotes—in the dark, this isozyme operates in glycolysis by oxidizing glyceraldehyde 3-phosphate. Both glyceraldehyde 3-phosphate dehydrogenase isozymes, like all enzymes, catalyze the reaction in both directions.

Fig. 20-14. Stoichiometry of СO₂ assimilation in the Calvin cycle. For every three fixed molecules of СO₂, one molecule of triose phosphate (glyceraldehyde 3-phosphate) is produced, while nine molecules of ATP and six molecules of NADPH are consumed.

One molecule of glyceraldehyde 3-phosphate is the net product of the carbon assimilation pathway. The other five molecules of triose phosphate (15 carbon atoms) undergo rearrangement (depicted in stages (1)–(9) of Fig. 20-10) to yield three molecules of ribulose 1,5-bisphosphate (15 carbon atoms). The final stage requires one molecule of ATP per molecule of ribulose 1,5-bisphosphate, totaling 3 ATP. Consequently, for every molecule of triose phosphate obtained through photosynthetic СO₂ assimilation, six NADPH and nine ATP are required.

NADPH and ATP are produced in the light-dependent reactions of photosynthesis in approximately the same ratio (2:3) in which they are consumed in the Calvin cycle. Nine molecules of ATP are converted into ADP and phosphate during the synthesis of a triose phosphate molecule; eight phosphate groups are released as P_i and condense with eight molecules of ADP, regenerating ATP. The ninth phosphate is incorporated into the triose phosphate. As we will see later, for the ninth ADP molecule to be converted into ATP, phosphate must be imported from the cytosol.

In the dark, the synthesis of ATP and NADPH via Photophosphorylation, as well as the incorporation of СO₂ into triose phosphates (the so-called dark reactions), ceases. The "dark reactions" of photosynthesis were originally named to distinguish them from the primary light-induced reactions of electron transfer to NADP⁺ and ATP synthesis (see Chapter 19). In reality, their rates are negligible in the dark; therefore, a more appropriate term for them is carbon assimilation reactions. Later in this section, we will describe The regulatory mechanisms that turn carbon assimilation on in the light and off in the dark.

The chloroplast stroma contains all the enzymes required to convert triose phosphates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate) derived from СO₂ assimilation into starch, which is temporarily stored in the chloroplast as insoluble granules. Aldolase condenses trioses into fructose 1,6-bisphosphate; fructose 1,6-bisphosphatase synthesizes fructose 6-phosphate; phosphohexose isomerase generates glucose 6-phosphate; and phosphoglucomutase produces glucose 1-phosphate, the starting material for starch synthesis (see Section 20.3).

All Reactions of the Calvin cycle, except those catalyzed by rubisco, sedoheptulose 1,7-bisphosphatase, and ribulose 5-phosphate kinase, also occur in animal tissues. However, without these three enzymes, animals cannot carry out the pathway converting СO₂ into glucose.

A transport system exports triose phosphates from the chloroplast and imports phosphate

The inner chloroplast membrane is impermeable to many phosphorylated compounds, including fructose 6-phosphate, glucose 6-phosphate, and fructose 1,6-bisphosphate. However, it contains a specific antiporter that catalyzes the exchange of P_i for the triose phosphate dihydroxyacetone phosphate or phosphoglycerate (Fig. 20-15, see also Fig. 20-9). This antiporter simultaneously transports phosphate into the chloroplast, where it is used in photophosphorylation, and triose phosphate into the cytosol, where it can be used for sucrose synthesis (it is in this form that fixed carbon is transported to distant plant tissues).

Fig. 20-15. The P_i-triose phosphate antiporter System of the inner chloroplast membrane. This transporter facilitates the exchange of cytosolic P_i for stromal dihydroxyacetone phosphate. The products of photosynthetic carbon assimilation move into the cytosol, where they serve as precursors for sucrose biosynthesis, while the P_i required for photophosphorylation moves into the stroma. This same antiporter can also transport 3-phosphoglycerate and participate in the export of ATP and reducing equivalents (see Fig. 20-16).

Cytosolic sucrose synthesis and chloroplast starch synthesis are the primary pathways that consume the excess triose phosphate produced during photosynthesis. Sucrose synthesis (described below) releases four P_i phosphates from the four triose phosphates required to synthesize a single sucrose molecule. For each molecule of triose phosphate exported, one P_i phosphate is transported into the chloroplast, supplying the ninth P_i needed for ATP regeneration (as mentioned earlier). If this exchange is blocked, triose phosphate synthesis rapidly depletes the available phosphate within the chloroplast, slowing down ATP synthesis and suppressing CO₂ incorporation into starch.

This P_i-triose phosphate antiporter system serves an additional function. Various synthetic and energy-requiring reactions in the cytosol demand ATP and reducing equivalents. While the extent to which Mitochondria replenish this pool remains unclear, a second potential energy resource consists of the ATP and NADPH produced by light reactions in the chloroplast stroma. However, neither ATP nor NADPH can cross the chloroplast membrane. The P_i-triose phosphate antiporter system overcomes this limitation by facilitating the export of ATP and reducing equivalents from the chloroplast into the cytosol (Fig. 20-16). Dihydroxyacetone phosphate synthesized in the stroma is transported to the cytosol, where glycolytic enzymes convert it to 3-phosphoglycerate, generating ATP and NADH in the process. The 3-phosphoglycerate then returns to the chloroplast, completing the cycle.

Fig. 20-16. Role of the P_i-triose phosphate antiporter in the Transport of ATP and reducing equivalents. Dihydroxyacetone phosphate leaves the chloroplast and is converted to glyceraldehyde 3-phosphate in the cytosol. Subsequent cytosolic glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase reactions yield NADH, ATP, and 3-phosphoglycerate. The return of 3-phosphoglycerate to the chloroplast and its reduction back to dihydroxyacetone phosphate complete a cycle that efficiently shuttles ATP and reducing equivalents (NADH/NADPH) from the chloroplast to the cytosol.

Four Calvin cycle enzymes are indirectly activated by light

The reductive assimilation of CO₂ requires substantial amounts of ATP and NADPH, and illumination of chloroplasts increases their concentration in the stroma (Fig. 20-17). Light-driven proton Transport Across the thylakoid membrane (Chap. 19) also raises the stromal pH from 7 to ~8, which is accompanied by the efflux of Mg²⁺ from the thylakoid lumen into the stroma, raising the Mg²⁺ concentration from 1–3 mM to 3–6 mM. Over the course of evolution, certain stromal enzymes have become adapted for activation upon illumination, when ATP and NADPH are readily available. These enzymes also exhibit higher activity in alkaline environments and at elevated Mg²⁺ concentrations. For instance, the activation of Rubisco via carbamoyllysine formation occurs more rapidly at alkaline pH, whereas high stromal Mg²⁺ concentrations favor

The formation of the catalytically active magnesium complex. Fructose-1,6-bisphosphatase requires Mg²⁺ and is highly pH-dependent (Fig. 20-18); its activity increases more than 100-fold as the pH and Mg²⁺ concentration rise upon chloroplast illumination.

Fig. 20-17. Sources of ATP and NADPH. ATP and NADPH generated in the light reactions serve as essential substrates for CO₂ reduction. Photosynthetic reactions producing ATP and NADPH are accompanied by proton translocation (shown in red) from the stroma into the thylakoid, creating alkaline conditions in the stroma. Magnesium ions pass from the thylakoid into the stroma, increasing the stromal Mg²⁺ concentration.

Fig. 20-18. Activation of chloroplast fructose-1,6-bisphosphatase. Inactive fructose-1,6-bisphosphatase (FBPase-1) is activated by light in response to the high pH and elevated stromal Mg²⁺ concentration that develop upon illumination.

Four Calvin cycle enzymes are subject to a specialized type of light regulation. Ribulose-5-phosphate kinase, fructose-1,6-bisphosphatase, sedoheptulose-1,7-bisphosphatase, and glyceraldehyde-3-phosphate dehydrogenase are activated upon illumination via the reduction of disulfide bonds between specific Cys residues, a modification required for their catalytic activity. When these Cys residues are disulfide-linked (oxidized), the enzymes are inactive—a state typical of darkness. Upon illumination, electrons are transferred from photosystem I to ferredoxin (see Fig. 19-56), which then passes them to a small, water-soluble, disulfide-containing protein called thioredoxin (Fig. 20-19) in a reaction catalyzed by ferredoxin-thioredoxin reductase. Reduced thioredoxin transfers electrons to the disulfide bonds of the light-activated enzymes; these bond-cleaving reduction reactions are coupled to conformational changes that enhance enzymatic activity. At dusk, the Cys residues of these four enzymes are reoxidized to their disulfide forms, inactivating the enzymes and preventing the wasteful consumption of ATP during CO₂ assimilation. Instead, starch synthesized and stored during the daytime is broken down into monomers to fuel glycolysis at night.

Fig. 20-19. Light activation of selected Calvin cycle enzymes. Light activation is mediated by thioredoxin, a small disulfide-containing protein. In the light, thioredoxin is reduced by electrons flowing from photosystem I through ferredoxin (Fd) (blue arrows); thioredoxin then reduces key disulfide bonds in sedoheptulose-1,7-bisphosphatase, fructose-1,6-bisphosphatase, ribulose-5-phosphate kinase, and glyceraldehyde-3-phosphate dehydrogenase, thereby activating them. In the dark, the -SH groups are reoxidized to disulfide bonds, inactivating the enzymes.

Glucose-6-phosphate dehydrogenase, the initial enzyme of the oxidative pentose phosphate pathway, is also regulated by this light-driven mechanism, but in the opposite direction. During the day, when photosynthesis generates abundant NADPH, this enzyme is not required for NADPH production. The reduction of a key disulfide bond by electrons from ferredoxin inactivates the enzyme.

Summary of Section 20.1 Photosynthesis of Carbohydrates

■ In vascular plants, photosynthesis takes place within chloroplasts. During CO₂ assimilation reactions (the Calvin cycle), ATP and NADPH are utilized to reduce CO₂ to triose phosphates. These reactions proceed in three stages: the fixation reaction itself, catalyzed by Rubisco; the reduction of the resulting 3-phosphoglycerate to glyceraldehyde 3-phosphate; and the regeneration of ribulose-1,5-bisphosphate from triose phosphates.

■ Rubisco condenses CO₂ with ribulose-1,5-bisphosphate to form an unstable hexose bisphosphate, which promptly 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 naturally occurring transition-state analog whose concentration rises in the dark and declines during the day.

■ Stromal isozymes of glycolytic enzymes catalyze the reduction of 3-phosphoglycerate to glyceraldehyde 3-phosphate, consuming one molecule of ATP and one molecule of NADPH per 3-phosphoglycerate molecule reduced.

■ Stromal enzymes, including transketolase and transaldolase, rearrange the carbon skeletons of triose phosphates to synthesize three-, four-, five-, six-, and seven-carbon intermediates, ultimately yielding pentose phosphates. These pentose phosphates are converted into ribulose-5-phosphate, which is subsequently phosphorylated to ribulose-1,5-bisphosphate, completing the Calvin cycle.

■ The "cost" of fixing three molecules of CO₂ into triose phosphate is nine molecules of ATP and six molecules of NADPH, which are synthesized during the light reactions of photosynthesis.

■ An antiporter located in the inner chloroplast membrane exchanges cytosolic P_i for the 3-phosphoglycerate or dihydroxyacetone phosphate produced in the stroma during CO₂ assimilation. Subsequent cytosolic oxidation of dihydroxyacetone phosphate yields ATP and NADH, effectively exporting ATP and reducing equivalents from the chloroplast to the cytosol.

■ Four Calvin cycle enzymes are indirectly activated by light and remain inactive in the dark, ensuring that hexose synthesis does not futilely compete with glycolysis, which is essential for energy production in the dark.