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

PART II. BIOENERGETICS AND METABOLISM

20. BIOSYNTHESIS OF CARBOHYDRATES IN PLANTS AND BACTERIA

20.3. Biosynthesis of Starch and Sucrose

During active Photosynthesis in bright light, plant leaves synthesize more CARBOHYDRATES (triose phosphates) than are required to meet their energy needs or to provide precursors for biosynthetic reactions. The excess is converted into sucrose and transported to other PARTS OF THE plant to serve as fuel or for storage. In most plants, starch is the primary reserve carbohydrate, but in some species, such as sugar beets and sugar cane, sucrose serves as the principal storage form. The synthesis of sucrose and starch takes place in different cellular compartments (the Cytosol and Plastids, respectively), and these processes are governed by distinct regulatory mechanisms that respond to changes in light intensity and The rate of photosynthesis.

ADP-Glucose is the Substrate for Starch Synthesis in Plant Plastids and Glycogen Synthesis in Bacteria

Starch, much like glycogen, is a high-molecular-weight polymer of D-glucose linked by α(1—>4) bonds. As one of the stable End products of photosynthesis, it is synthesized for temporary storage within METABOLISM/14.html">Chloroplasts, whereas for long-term reserve storage it is produced in non-photosynthetic plant Tissues—seeds, roots, and tubers (underground stems).

The mechanism by which glucose is incorporated into starch closely parallels that of glycogen synthesis. The Condensation of glucose-1-phosphate with ATP—in a reaction driven far to the right by the presence of inorganic pyrophosphatase (p. 38)—yields an activated sugar nucleotide, in this case ADP-glucose. Starch synthase then transfers a glucose residue from ADP-glucose to a pre-existing starch molecule. Although it was traditionally believed that glucose is added to the nonreducing end of starch, much like in glycogen synthesis (see Fig. 15-30), recent evidence indicates that starch synthase possesses two equivalent active sites that alternately add glycosyl units to the reducing end of the starch chain. This end remains covalently bound to the enzyme, alternating between one Active Site and the other (Fig. 20-24). The addition of a glycosyl moiety to one active site effectively activates the reducing end of the growing starch chain. This is followed by a nucleophilic Cleavage from the enzyme via an attack by the hydroxyl group at the C-4 atom of the glucose residue bound at the second active site, thereby forming the characteristic α(1—>4) linkage of starch.

Class="center">Fig. 20-24. Starch synthesis. According to this model, starch synthesis involves two active sites and utilizes ADP-glucose as the glycosyl donor. Two identical active sites in starch synthase alternately add glucose units to the growing chain at the reducing end.

The amylose component of starch is unbranched, whereas amylopectin contains numerous α(1—>6)-linked branch points (see Fig. 7-14). Chloroplasts contain a branching enzyme, analogous to the glycogen-branching enzyme (see Fig. 15-31), which introduces the α(1—>6) branches into amylopectin. Taking into account the Hydrolysis of pyrophosphate (PP_i) released during the synthesis of ADP-glucose by inorganic pyrophosphatase, the overall reaction for The formation of starch from glucose-1-phosphate can be written as follows:

Starch_n + glucose-1-phosphate + ATP —> starch_n+1 + ADP + 2P_i

∆G'° = -50 kJ/mol

Starch synthesis is regulated primarily by the rate of ADP-glucose formation, as discussed below.

Most bacteria store carbohydrates in the form of glycogen (which resembles starch, but is more extensively branched), synthesized via a pathway analogous to the glycogen synthase reaction in animals. Like plant plastids, bacteria utilize ADP-glucose as the activated form of glucose, whereas animal Cells use UDP-glucose. Once again, this metabolic similarity between plastids and bacteria provides strong support for the Endosymbiotic Hypothesis of organelle evolution (see p. 59, Vol. 1).

UDP-Glucose is the Substrate for Sucrose Synthesis in the Cytosol of Leaf Cells

The bulk of the triose phosphates produced during photosynthetic CO₂ fixation is converted into sucrose (Fig. 20-25) or starch. Over the course of evolution, sucrose may have been selected as the primary carbon transport molecule because of the unusual linkage in its molecule between the anomeric C-1 atom of glucose and the anomeric C-2 atom of fructose. This bond is not attacked by amylases or other common carbohydrate-cleaving Enzymes, and the inaccessibility of the anomeric carbon atoms prevents non-ENZYMATIC REACTIONS OF sucrose with Amino Acids and Proteins (reactions to which glucose is prone).

Fig. 20-25. Sucrose synthesis. Sucrose is synthesized from UDP-glucose and fructose-6-phosphate, which are derived from triose phosphates in the plant cytosol via the pathways shown in Figures 15-29 and 20-9. In most plant species, sucrose-6-phosphate synthase is allosterically regulated by glucose-6-phosphate and inorganic phosphate (P_i).

Sucrose synthesis takes place in the cytosol and begins with The transport of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate out of the chloroplasts. Following the condensation of two triose phosphates to yield fructose-1,6-bisphosphate (catalyzed by aldolase), fructose-1,6-bisphosphate is hydrolyzed to form fructose-6-phosphate. Next, sucrose-6-phosphate synthase catalyzes the reaction of fructose-6-phosphate with UDP-glucose to produce sucrose-6-phosphate (Fig. 20-25). Finally, sucrose-6-phosphate phosphatase removes the phosphate group, yielding free sucrose ready for export to other tissues. The reaction catalyzed by sucrose-6-phosphate synthase is a low-energy process (∆G′° = -5.7 kJ/mol), but the subsequent hydrolysis of sucrose-6-phosphate to sucrose is sufficiently exothermic (∆G′° = -16.5 kJ/mol) to render the overall pathway of sucrose synthesis essentially irreversible. Sucrose synthesis is tightly regulated and closely coordinated with starch synthesis, as we shall see later.

One notable difference between PLANT AND ANIMAL cells lies in the absence within the plant cytosol of inorganic pyrophosphatase, an enzyme that catalyzes the reaction:

PP_i + H₂O —> 2P_i ∆G′° = -19.2 kJ/mol

For many biosynthetic reactions that release pyrophosphate (PP_i), pyrophosphatase activity pulls the reaction forward, making it energetically more favorable and ensuring its irreversibility. In plants, this enzyme is present in plastids but absent from the cytosol. As a consequence, the cytosol of leaf cells maintains a significant concentration of pyrophosphate (~0.3 mM), which keeps reactions such as the one catalyzed by UDP-glucose pyrophosphorylase (Fig. 15-29) readily reversible. As we recall from Chapter 14 (p. 73), the cytosolic isozyme of Phosphofructokinase in plants utilizes PP_i rather than ATP as its phosphate donor.

The Partitioning of Triose Phosphates into Sucrose and Starch is Strictly Regulated

As noted previously, the triose phosphates generated by The Calvin Cycle in bright daylight can be temporarily stored as starch within the chloroplasts, converted into sucrose for export to non-photosynthetic plant tissues, or both. The balance between these two pathways is rigorously regulated and coordinated with the rate of carbon fixation. Five out of every six triose phosphate molecules produced in the Calvin cycle must be recycled to regenerate ribulose-1,5-bisphosphate (Fig. 20-14); if more than one out of every six triose phosphate molecules is drawn off from the cycle for sucrose or starch synthesis, the cycle will slow down or stall. Conversely, inadequate conversion of triose phosphates into starch or sucrose occurs when a P_i-depleted chloroplast is starved for the phosphate required to sustain the Calvin cycle.

The rate at which triose phosphates are incorporated into sucrose is regulated by The activity of fructose-1,6-bisphosphatase (FBPase-1) and an enzyme that catalyzes the opposing reaction—pyrophosphate-dependent phosphofructokinase (PPi-PFK-1; p. 73). These enzymes play a pivotal role in determining the metabolic fate of triose phosphates generated during photosynthesis. Both enzymes are regulated by fructose-2,6-bisphosphate (F2,6BP), which inhibits FBPase-1 and stimulates PPi-PFK-1. In vascular plants, the concentration of F2,6BP is inversely proportional to the rate of photosynthesis (Fig. 20-26). Phosphofructokinase-2 (PFK-2), the enzyme responsible for F2,6BP synthesis, is inhibited by dihydroxyacetone phosphate or 3-phosphoglycerate and stimulated by fructose-6-phosphate and P_i. During active photosynthesis, the concentration of dihydroxyacetone phosphate increases while the concentration of P_i decreases, resulting in the inhibition of PFK-2 and a drop in F2,6BP levels. This promotes a higher rate of triose phosphate conversion into fructose-6-phosphate and, subsequently, sucrose. Through this regulatory circuit, sucrose synthesis occurs when the pool of triose phosphates produced in the Calvin cycle significantly exceeds the level required to sustain cycle operation.

Sucrose synthesis is also regulated by the levels of sucrose-6-phosphate synthase, which is allosterically activated by glucose-6-phosphate and inhibited by inorganic phosphate (P_i). This enzyme is further modulated by phosphorylation and dephosphorylation: a protein kinase phosphorylates a specific Ser residue, rendering sucrose-6-phosphate synthase less active, whereas a phosphatase reverses this inhibition by removing the phosphate group (Fig. 20-27). The inhibition of the kinase by glucose-6-phosphate and the phosphatase by inorganic phosphate reinforces the coordinated control of these two compounds over sucrose synthesis. When hexose phosphates accumulate in excess, sucrose-6-phosphate synthase is activated by glucose-6-phosphate; conversely, when the level of P_i rises (signaling a decline in the rate of photosynthesis), sucrose synthesis slows down. During active photosynthesis, triose phosphates are converted into fructose-6-phosphate, which is rapidly isomerized to glucose-6-phosphate in a reversible reaction catalyzed by phosphoglucoisomerase. Because the equilibrium heavily favors glucose-6-phosphate, any accumulation of fructose-6-phosphate drives up glucose-6-phosphate levels, thereby stimulating sucrose synthesis.

Fig. 20-26. Fructose-2,6-bisphosphate as a regulator of sucrose synthesis. The concentration of the allosteric regulator fructose-2,6-bisphosphate within plant cells is governed by the products of photosynthetic carbon assimilation and P_i. Dihydroxyacetone phosphate and 3-phosphoglycerate, produced during CO₂ assimilation, inhibit phosphofructokinase-2 (PFK-2)—the enzyme that synthesizes the regulator—whereas P_i stimulates PFK-2. Consequently, the concentration of the regulator varies inversely with the rate of photosynthesis. In the dark, the concentration of fructose-2,6-bisphosphate rises, which stimulates the glycolytic enzyme PPi-dependent phosphofructokinase-1 (PPi-PFK-1) while inhibiting the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase-1). During active photosynthesis (in the light), the concentration of the regulator drops, thereby stimulating the synthesis of fructose-6-phosphate and sucrose.

Fig. 20-27. Regulation of sucrose-phosphate synthase by phosphorylation. A protein kinase specific for sucrose-phosphate synthase (SPS kinase) phosphorylates a specific Ser residue on SPS, thereby inactivating it, whereas a specific phosphatase (SPS phosphatase) removes this inhibitory group. The kinase is allosterically inhibited by glucose-6-phosphate, a compound that simultaneously acts as an allosteric activator of SPS. The phosphatase is inhibited by P_i, which also directly inhibits SPS. Thus, when active photosynthesis yields a high concentration of glucose-6-phosphate, SPS is activated to synthesize sucrose phosphate. Conversely, elevated concentrations of P_i—which accumulate when the rate of ADP conversion to ATP during photosynthesis drops—inhibit sucrose phosphate synthesis.

The key regulatory enzyme in starch Biosynthesis is ADP-glucose pyrophosphorylase (Fig. 20-28); it is activated by 3-phosphoglycerate (which accumulates during active photosynthesis) and inhibited by inorganic phosphate P_i (which builds up when the light-driven condensation of phosphate with ADP diminishes). When sucrose synthesis slows down, the 3-phosphoglycerate generated via CO₂ fixation accumulates, activating the enzyme and driving starch synthesis.

Fig. 20-28. Regulation of ADP-glucose pyrophosphorylase by 3-phosphoglycerate and P_i. This enzyme synthesizes the starch precursor and serves as the rate-limiting step in starch biosynthesis. It is allosterically stimulated by 3-phosphoglycerate (3-PGA) and inhibited by P_i; the ratio [3-PGA]/[P_i], which increases as photosynthetic rates rise, is a critical determinant for this stage of starch formation.

Summary of Section 20.3 Biosynthesis of Starch and Sucrose

■ Starch synthase in chloroplasts and amyloplasts catalyzes the sequential addition of glucose residues from ADP-glucose to the non-reducing end of a growing polysaccharide chain via two active sites. A second enzyme is responsible for amylopectin branching.

■ Sucrose is synthesized in the cytosol from UDP-glucose and fructose-6-phosphate in a two-step process.

■ The partitioning of triose phosphates between sucrose and starch synthesis is regulated by fructose-2,6-bisphosphate (F2,6BP), an allosteric effector of the enzyme that controls fructose-6-phosphate levels. The concentration of F2,6BP is inversely proportional to the rate of photosynthesis, and F2,6BP inhibits the Formation of fructose-6-phosphate, the precursor to sucrose.