Bread and pasteries on a rack at a French bakery,
Paris. Carbohydrates such as these provide a significant
portion of human caloric intake. (Ó Steven Rothfeld/Tony
Stone Images)

Chapter 23

Gluconeogenesis, Glycogen Metabolism, and the Pentose Phosphate Pathway

As shown in Chapters 18 and 19, the metabolism of sugars is an important source of energy for cells. Animals, including humans, typically obtain significant amounts of glucose and other sugars from the breakdown of starch and glycogen in their diets. Glucose can also be supplied via breakdown of cellular reserves of glycogen (in animals) or starch (in plants). Significantly, glucose also can be synthesized from noncarbohydrate precursors by a process known as gluconeogenesis. Each of these important pathways, as well as the synthesis of glycogen from glucose, will be examined in this chapter.
      Another pathway of glucose catabolism, the pentose phosphate pathway, is the primary source of NADPH, the reduced coenzyme essential to most reductive biosynthetic processes. For example, NADPH is crucial to the biosynthesis of fatty acids (Chapter 25) and amino acids (Chapter 26). The pentose phosphate pathway also results in the production of ribose-5-phosphate, an essential component of ATP, NAD⁺, FAD, coenzyme A, and particularly DNA and RNA. This important pathway will also be considered in this chapter.

23.1 · Gluconeogenesis

The ability to synthesize glucose from common metabolites is very important to most organisms. Human metabolism, for example, consumes about 160 ± 20 grams of glucose per day, about 75% of this in the brain. Body fluids carry only about 20 grams of free glucose, and glycogen stores normally can provide only about 180 to 200 grams of free glucose. Thus, the body carries only a little more than a one-day supply of glucose. If glucose is not obtained in the diet, the body must produce new glucose from noncarbohydrate precursors. The term for this activity is gluconeogenesis, which means the generation (genesis) of new (neo) glucose.
      Further, muscles consume large amounts of glucose via glycolysis, producing large amounts of pyruvate. In vigorous exercise, muscle cells become anaerobic and pyruvate is converted to lactate. Gluconeogenesis salvages this pyruvate and lactate and reconverts it to glucose.

The Substrates of Gluconeogenesis

In addition to pyruvate and lactate, other noncarbohydrate precursors can be used as substrates for gluconeogenesis in animals. These include most of the amino acids, as well as glycerol and all the TCA cycle intermediates. On the other hand, fatty acids are not substrates for gluconeogenesis in animals, because most fatty acids yield only acetyl-CoA upon degradation, and animals cannot carry out net synthesis of sugars from acetyl-CoA. Lysine and leucine are the only amino acids that are not substrates for gluconeogenesis. These amino acids produce only acetyl-CoA upon degradation. Note also that acetyl-CoA is a substrate for gluconeogenesis when the glyoxylate cycle is operating (Chapter 20).

Nearly All Gluconeogenesis Occurs in the Liver and Kidneys in Animals

Interestingly, the mammalian organs that consume the most glucose, namely, brain and muscle, carry out very little glucose synthesis. The major sites of gluconeogenesis are the liver and kidneys, which account for about 90% and 10% of the body’s gluconeogenic activity, respectively. Glucose produced by gluconeogenesis in the liver and kidney is released into the blood and is subsequently absorbed by brain, heart, muscle, and red blood cells to meet their metabolic needs. In turn, pyruvate and lactate produced in these tissues are returned to the liver and kidney to be used as gluconeogenic substrates.

Gluconeogenesis Is Not Merely the Reverse of Glycolysis

In some ways, gluconeogenesis is the reverse, or antithesis, of glycolysis. Glucose is synthesized, not catabolized; ATP is consumed, not produced; and NADH is oxidized to NAD⁺, rather than the other way around. However, gluconeogenesis cannot be merely the reversal of glycolysis, for two reasons. First, glycolysis is exergonic, with a DG°' of approximately -74 kJ/mol. If gluconeogenesis were merely the reverse, it would be a strongly endergonic process and could not occur spontaneously. Somehow the energetics of the process must be augmented so that gluconeogenesis can proceed spontaneously. Second, the processes of glycolysis and gluconeogenesis must be regulated in a reciprocal fashion so that when glycolysis is active, gluconeogenesis is inhibited, and when gluconeogenesis is proceeding, glycolysis is turned off. Both of these limitations are overcome by having unique reactions within the routes of glycolysis and gluconeogenesis, rather than a completely shared pathway.

Figure 23.1 · The pathways of gluconeo-genesis and glycolysis. Species in blue, green, and peach-colored shaded boxes indicate other entry points for gluconeogenesis (in addition to pyruvate).

Gluconeogenesis—Something Borrowed, Something New

The complete route of gluconeogenesis is shown in Figure 23.1, side by side with the glycolytic pathway. Gluconeogenesis employs three different reactions, catalyzed by three different enzymes, for the three steps of glycolysis that are highly exergonic (and highly regulated). In essence, seven of the ten steps of glycolysis are merely reversed in gluconeogenesis. The six reactions between fructose-1,6-bisphosphate and PEP are shared by the two pathways, as is the isomerization of glucose-6-P to fructose-6-P. The three exergonic regulated reactions—the hexokinase (glucokinase), phosphofructokinase, and pyruvate kinase reactions—are replaced by alternative reactions in the gluconeogenic pathway.
      The conversion of pyruvate to PEP that initiates gluconeogenesis is accomplished by two unique reactions. Pyruvate carboxylase catalyzes the first, converting pyruvate to oxaloacetate. Then, PEP carboxykinase catalyzes the conversion of oxaloacetate to PEP. Conversion of fructose-1,6-bisphosphate to fructose-6-phosphate is catalyzed by a specific phosphatase, fructose-1,6-bisphosphatase. The final step to produce glucose, hydrolysis of glucose-6-phosphate, is mediated by glucose-6-phosphatase. Each of these steps is considered in detail in the following paragraphs. The overall conversion of pyruvate to PEP by pyruvate carboxylase and PEP carboxykinase has a DG°' close to zero but is pulled along by subsequent reactions. The conversion of fructose-1,6-bisphosphate to glucose in the last three steps of gluconeogenesis is strongly exergonic with a DG°' of about -30.5 kJ/mol. This sequence of two phosphatase reactions separated by an isomerization accounts for most of the free energy release that makes the gluconeogenesis pathway spontaneous.

The Unique Reactions of Gluconeogenesis

(1) Pyruvate Carboxylase—A Biotin-Dependent Enzyme

Initiation of gluconeogenesis occurs in the pyruvate carboxylase reaction—the conversion of pyruvate to oxaloacetate (Figure 23.2). The reaction takes place in two discrete steps, involves ATP and bicarbonate as substrates, and utilizes biotin as a coenzyme and acetyl-coenzyme A as an allosteric activator. Pyruvate carboxylase is a tetrameric enzyme (with a molecular mass of about 500 kD). Each monomer possesses a biotin covalently linked to the e-amino group of a lysine residue at the active site (Figure 23.3). The first step of the reaction involves nucleophilic attack of a bicarbonate oxygen at the g-P of ATP to form carbonylphosphate, an activated form of CO₂, and ADP (Figure 23.4). Reaction of carbonylphosphate with biotin occurs rapidly to form N-carboxybiotin, liberating inorganic phosphate. The third step involves abstraction of a proton from the C-3 of pyruvate, forming a carbanion which can attack the carbon of N-carboxybiotin to form oxaloacetate.

Figure 23.2 · The pyruvate carboxylase reaction.

Figure 23.3 · Covalent linkage of biotin to an active-site lysine in pyruvate carboxylase.

Figure 23.4 · A mechanism for the pyruvate carboxylase reaction. Bicarbonate must be activated for attack by the pyruvate carbanion. This activation is driven by ATP and involves formation of a carbonylphosphate intermediate—a mixed anhydride of carbonic and phosphoric acids. (Carbonylphosphate and carboxyphosphate are synonyms.)

PYRUVATE CARBOXYLASE IS ALLOSTERICALLY ACTIVATED BY ACYL-COENZYME A  
Two particularly interesting aspects of the pyruvate carboxylase reaction are (a) allosteric activation of the enzyme by acyl-coenzyme A derivatives and (b) compartmentation of the reaction in the mitochondrial matrix. The carboxylation of biotin requires the presence (at an allosteric site) of acetyl-coenzyme A or other acylated coenzyme A derivatives. The second half of the carboxylase reaction—the attack by pyruvate to form oxaloacetate—is not affected by CoA derivatives.
      Activation of pyruvate carboxylase by acetyl-CoA provides an important physiological regulation. Acetyl-CoA is the primary substrate for the TCA cycle, and oxaloacetate (formed by pyruvate carboxylase) is an important intermediate in both the TCA cycle and the gluconeogenesis pathway. If levels of ATP and/or acetyl-CoA (or other acyl-CoAs) are low, pyruvate is directed primarily into the TCA cycle, which eventually promotes the synthesis of ATP. If ATP and acetyl-CoA levels are high, pyruvate is converted to oxaloacetate and consumed in gluconeogenesis. Clearly, high levels of ATP and CoA derivatives are signs that energy is abundant and that metabolites will be converted to glucose (and perhaps even glycogen). If the energy status of the cell is low (in terms of ATP and CoA derivatives), pyruvate is consumed in the TCA cycle. Also, as noted in Chapter 20, pyruvate carboxylase is an important anaplerotic enzyme. Its activation by acetyl-CoA leads to oxaloacetate formation, replenishing the level of TCA cycle intermediates.

COMPARTMENTALIZED PYRUVATE CARBOXYLASE DEPENDS ON METABOLITE CONVERSION AND TRANSPORT      The second interesting feature of pyruvate carboxylase is that it is found only in the matrix of the mitochondria. By contrast, the next enzyme in the gluconeogenic pathway, PEP carboxykinase, may be localized in the cytosol or in the mitochondria or both. For example, rabbit liver PEP carboxykinase is predominantly mitochondrial, whereas the rat liver enzyme is strictly cytosolic. In human liver, PEP carboxykinase is found both in the cytosol and in the mitochondria. Pyruvate is transported into the mitochondrial matrix, where it can be converted to acetyl-CoA (for use in the TCA cycle) and then to citrate (for fatty acid synthesis; see Figure 25.1). Alternatively, it may be converted directly to OAA by pyruvate carboxylase and used in gluconeogenesis. In tissues where PEP carboxykinase is found only in the mitochondria, oxaloacetate is converted to PEP, which is then transported to the cytosol for gluconeogenesis (Figure 23.6). However, in tissues that must convert some oxaloacetate to PEP in the cytosol, a problem arises. Oxaloacetate cannot be transported directly across the mitochondrial membrane. Instead, it must first be transformed into malate or aspartate for transport across the mitochondrial inner membrane (Figure 23.5). Cytosolic malate and aspartate must be reconverted to oxaloacetate before continuing along the gluconeogenic route.

Figure 23.5 · Pyruvate carboxylase is a compartmentalized reaction. Pyruvate is converted to oxaloacetate in the mitochondria. Because oxaloacetate cannot be transported across the mitochondrial membrane, it must be reduced to malate, transported to the cytosol, and then oxidized back to oxaloacetate before gluconeogenesis can continue.

(2) PEP Carboxykinase

The second reaction in the gluconeogenic pyruvate-PEP bypass is the conversion of oxaloacetate to PEP. Production of a high-energy metabolite such as PEP requires energy. The energetic requirements are handled in two ways here. First, the CO₂ added to pyruvate in the pyruvate carboxylase step is removed in the PEP carboxykinase reaction. Decarboxylation is a favorable process and helps to drive the formation of the very high-energy enol phosphate in PEP. This decarboxylation drives a reaction that would otherwise be highly endergonic. Note the inherent metabolic logic in this pair of reactions: pyruvate carboxylase consumed an ATP to drive a carboxylation, so that the PEP carboxykinase could use the decarboxylation to facilitate formation of PEP. Second, as shown in Figure 23.6, another high-energy phosphate is consumed by the carboxykinase. Mammals and several other species use GTP in this reaction, rather than ATP. The use of GTP here is equivalent to the consumption of an ATP, due to the activity of the nucleoside diphosphate kinase (see Figure 20.4). The substantial free energy of hydrolysis of GTP is crucial to the synthesis of PEP in this step. The overall DG for the pyruvate carboxylase and PEP carboxykinase reactions under physiological conditions in the liver is -22.6 kJ/mol. Once PEP is formed in this way, the phosphoglycerate mutase, phosphoglycerate kinase, glyceraldehyde-3-P dehydrogenase, aldolase, and triose phosphate isomerase reactions act to eventually form fructose-1,6-bisphosphate, as in Figure 23.1.

Figure 23.6 · The PEP carboxykinase reaction. GTP formed in this reaction can be converted to ATP by nucleoside diphosphate kinase, although liver cells in some species may not contain this enzyme.

(3) Fructose-1,6-Bisphosphatase

The hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate (Figure 23.7), like all phosphate ester hydrolyses, is a thermodynamically favorable (exergonic) reaction under standard-state conditions (DG°' = -16.7 kJ/mol). Under physiological conditions in the liver, the reaction is also exergonic (DG = -8.6 kJ/mol). Fructose-1,6-bisphosphatase is an allosterically regulated enzyme. Citrate stimulates bisphosphatase activity, but fructose-2,6-bisphosphate is a potent allosteric inhibitor. AMP also inhibits the bisphosphatase; the inhibition by AMP is enhanced by fructose-2,6-bisphosphate.

Figure 23.7 · The fructose-1,6-bisphosphatase reaction.

(4) Glucose-6-Phosphatase

The final step in the gluconeogenesis pathway is the conversion of glucose-6-phosphate to glucose by the action of glucose-6-phosphatase. This enzyme is present in the membranes of the endoplasmic reticulum of liver and kidney cells, but is absent in muscle and brain. For this reason, gluconeogenesis is not carried out in muscle and brain. Its membrane association is important to its function because (Figure 23.8) the substrate is hydrolyzed as it passes into the endoplasmic reticulum itself. Vesicles form from the endoplasmic reticulum membrane and diffuse to the plasma membrane and fuse with it, releasing their glucose contents into the bloodstream. The glucose-6-phosphatase reaction involves a phosphorylated enzyme intermediate, which may be a phosphohistidine (Figure 23.9). The DG for the glucose-6-phosphatase reaction in liver is -5.1 kJ/mol.

Figure 23.8 · Glucose-6-phosphatase is localized in the endoplasmic reticulum membrane. Conversion of glucose-6-phosphate to glucose occurs during transport into the ER.

Figure 23.9 · The glucose-6-phosphatase reaction involves formation of a phosphohistidine intermediate.

COUPLING WITH HYDROLYSIS OF ATP AND GTP DRIVES GLUCONEOGENESIS      The net reaction for the conversion of pyruvate to glucose in gluconeogenesis is

          2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H⁺ + 6 H₂O
                                                  ¯
                    glucose + 4 ADP + 2 GDP + 6 P_i + 2 NAD⁺

The net free energy change, DG°', for this conversion is -37.7 kJ/mol. The consumption of a total of six nucleoside triphosphates drives this process forward. If glycolysis were merely reversed to achieve the net synthesis of glucose from pyruvate, the net reaction would be

                        2 Pyruvate+2 ATP+2 NADH+2 H⁺+2 H₂O
                                                       ¯
                                      glucose+2 ADP+2 P_i+2 NAD⁺

and the overall DG°' would be about +74 kJ/mol. Such a process would be highly endergonic, and therefore thermodynamically unfeasible. Hydrolysis of four additional high-energy phosphate bonds makes gluconeogenesis thermodynamically favorable. Under physiological conditions, however, gluconeogenesis is somewhat less favorable than at standard state, with an overall DG of -15.6 kJ/mol for the conversion of pyruvate to glucose.

Figure 23.10 · The Cori cycle.

LACTATE FORMED IN MUSCLES IS RECYCLED TO GLUCOSE IN THE LIVER      A final point on the redistribution of lactate and glucose in the body serves to emphasize the metabolic interactions between organs. Vigorous exercise can lead to oxygen shortage (anaerobic conditions), and energy requirements must be met by increased levels of glycolysis. Under such conditions, glycolysis converts NAD⁺ to NADH, yet O₂ is unavailable for regeneration of NAD⁺ via cellular respiration. Instead, large amounts of NADH are reoxidized by the reduction of pyruvate to lactate. The lactate thus produced can be transported from muscle to the liver, where it is reoxidized by liver lactate dehydrogenase to yield pyruvate, which is converted eventually to glucose. In this way, the liver shares in the metabolic stress created by vigorous exercise. It exports glucose to muscle, which produces lactate, which can be processed by the liver into new glucose. This is referred to as the Cori cycle (Figure 23.10). Liver, with a typically high NAD⁺/NADH ratio (about 700), readily produces more glucose than it can use. Muscle that is vigorously exercising will enter anaerobiosis and show a decreasing NAD⁺/NADH ratio, which favors reduction of pyruvate to lactate.

23.2 · Regulation of Gluconeogenesis

Nearly all of the reactions of glycolysis and gluconeogenesis take place in the cytosol. If metabolic control were not exerted over these reactions, glycolytic degradation of glucose and gluconeogenic synthesis of glucose could operate simultaneously, with no net benefit to the cell and with considerable consumption of ATP. This is prevented by a sophisticated system of reciprocal control, so that glycolysis is inhibited when gluconeogenesis is active, and vice versa. Reciprocal regulation of these two pathways depends largely on the energy status of the cell. When the energy status of the cell is low, glucose is rapidly degraded to produce needed energy. When the energy status is high, pyruvate and other metabolites are utilized for synthesis (and storage) of glucose.
      In glycolysis, the three regulated enzymes are those catalyzing the strongly exergonic reactions: hexokinase (glucokinase), phosphofructokinase, and pyruvate kinase. As noted, the gluconeogenic pathway replaces these three reactions with corresponding reactions that are exergonic in the direction of glucose synthesis: glucose-6-phosphatase, fructose-1,6-bisphosphatase, and the pyruvate carboxylase-PEP carboxykinase pair, respectively. These are the three most appropriate sites of regulation in gluconeogenesis.

Gluconeogenesis Is Regulated by Allosteric and Substrate-Level Control Mechanisms

The mechanisms of regulation of gluconeogenesis are shown in Figure 23.11. Control is exerted at all of the predicted sites, but in different ways. Glucose-6-phosphatase is not under allosteric control. However, the K_m for the substrate, glucose-6-phosphate, is considerably higher than the normal range of substrate concentrations. As a result, glucose-6-phosphatase displays a near-linear dependence of activity on substrate concentrations and is thus said to be under substrate-level control by glucose-6-phosphate.

Figure 23.11 · The principal regulatory mechanisms in glycolysis and gluconeogenesis. Activators are indicated by plus signs and inhibitors by minus signs.

      Acetyl-CoA is a potent allosteric effector of glycolysis and gluconeogenesis. It allosterically inhibits pyruvate kinase (as noted in Chapter 19) and activates pyruvate carboxylase. Because it also allosterically inhibits pyruvate dehydrogenase (the enzymatic link between glycolysis and the TCA cycle), the cellular fate of pyruvate is strongly dependent on acetyl-CoA levels. A rise in [acetyl-CoA] indicates that cellular energy levels are high and that carbon metabolites can be directed to glucose synthesis and storage. When acetyl-CoA levels drop, the activities of pyruvate kinase and pyruvate dehydrogenase increase and flux through the TCA cycle increases, providing needed energy for the cell.
      Fructose-1,6-bisphosphatase is another important site of gluconeogenic regulation. This enzyme is inhibited by AMP and activated by citrate. These effects by AMP and citrate are the opposites of those exerted on phosphofructokinase in glycolysis, providing another example of reciprocal regulatory effects. When AMP levels increase, gluconeogenic activity is diminished and glycolysis is stimulated. An increase in citrate concentration signals that TCA cycle activity can be curtailed and that pyruvate should be directed to sugar synthesis instead.

Fructose-2,6-Bisphosphate—Allosteric Regulator of Gluconeogenesis

As described in Chapter 19, Emile Van Schaftingen and Henri-Géry Hers demonstrated in 1980 that fructose-2,6-bisphosphate is apotent stimulator of phosphofructokinase. Cognizant of the reciprocal nature of regulation in glycolysis and gluconeogenesis, Van Schaftingen and Hers also considered the possibility of an opposite effect—inhibition—for fructose-1,6-bisphosphatase. In 1981 they reported that fructose-2,6-bisphosphate was indeed a powerful inhibitor of fructose-1,6-bisphosphatase (Figure 23.12). Inhibition occurs in either the presence or absence of AMP, and the effects of AMP and fructose-2,6-bisphosphate are synergistic.

Figure 23.12 · Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate in the (a) absence and (b) presence of 25 mM AMP. In (a) and (b), enzyme activity is plotted against substrate (fructose-1,6-bisphosphate) concentration. Concentrations of fructose-2,6-bisphosphate (in mM) are indicated above each curve. (c) The effect of AMP (0, 10, and 25 mM) on the inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphos-phate. Activity was measured in the presence of 10 mM fructose-1,6-bisphosphate. (Adapted from Van Schaftingen, E., and Hers, H.-G., 1981. Inhibition of fructose-1,6-bisphosphatase by fructose-2,6-bisphosphate.Proceedings of the National Academy of Science, USA 78:2861-2863.)

      Cellular levels of fructose-2,6-bisphosphate are controlled by phosphofructokinase-2 (PFK-2), an enzyme distinct from the phosphofructokinase of the glycolytic pathway, and by fructose-2,6-bisphosphatase (F-2,6-BPase). Remarkably, these two enzymatic activities are both found in the same protein molecule, which is an example of a bifunctional, or tandem, enzyme (Figure 23.13). The opposing activities of this bifunctional enzyme are themselves regulated in two ways. First, fructose-6-phosphate, the substrate of phosphofructokinase and the product of fructose-1,6-bisphosphatase, allosterically activates PFK-2 and inhibits F-2,6-BPase. Second, the phosphorylation by cAMP-dependent protein kinase of a single Ser residue on the 49-kD subunit of this dimeric enzyme exerts reciprocal control of the PFK-2 and F-2,6-BPase activities. Phosphorylation then inhibits PFK-2 activity (by increasing the K_m for fructose-6-phosphate) and stimulates F-2,6-BPase activity.

Figure 23.13 · Synthesis and degradation of fructose-2,6-bisphosphate are catalyzed by the same bifunctional enzyme.

Substrate Cycles Provide Metabolic Control Mechanisms

If fructose-1,6-bisphosphatase and phosphofructokinase acted simultaneously, they would constitute a substrate cycle in which fructose-1,6-bisphosphate and fructose-6-phosphate became interconverted with net consumption of ATP:

                       Fructose-1,6-bisP+H₂O ® fructose-6-P+P_i
                       Fructose-6-P+ATP ® fructose-1,6-bisP+ADP

Because substrate cycles such as this appear to operate with no net benefit to the cell, they were once regarded as metabolic quirks and were referred to as futile cycles. More recently, substrate cycles have been recognized as important devices for controlling metabolite concentrations.
      The three steps in glycolysis and gluconeogenesis that differ constitute three such substrate cycles, each with its own particular metabolic raison d’être. Consider, for example, the regulation of the fructose-1,6-bisP-fructose-6-P cycle by fructose-2,6-bisphosphate. As already noted, fructose-1,6-bisphosphatase is subject to allosteric inhibition by fructose-2,6-bisphosphate, whereas phosphofructokinase is allosterically activated by fructose-2,6-bisP. The combination of these effects should permit either phosphofructokinase or fructose-1,6-bisphosphatase (but not both) to operate at any one time and should thus prevent futile cycling. For instance, in the fasting state, when food (i.e., glucose) intake is zero, phosphofructokinase (and therefore glycolysis) is inactive due to the low concentration of fructose-2,6-bisphosphate. In the liver, gluconeogenesis operates to provide glucose for the brain. However, in the fed state, up to 30% of fructose-1,6-bisphosphate formed from phosphofructokinase is recycled back to fructose-6-P (and then to glucose). Because the dependence of fructose-1,6-bisphosphatase activity on fructose-1,6-bisphosphate is sigmoidal in the presence of fructose-2,6-bisphosphate (Figure 23.12), substrate cycling occurs only at relatively high levels of fructose-1,6-bisphosphate. Substrate cycling in this case prevents the accumulation of excessively high levels of fructose-1,6-bisphosphate.

23.3 · Glycogen Catabolism

Dietary Glycogen and Starch Breakdown

As noted earlier, well-fed adult human beings normally metabolize about 160 g of carbohydrates each day. A balanced diet easily provides this amount, mostly in the form of starch, with smaller amounts of glycogen. If too little carbohydrate is supplied by the diet, glycogen reserves in liver and muscle tissue can also be mobilized. The reactions by which ingested starch and glycogen are digested are shown in Figure 23.14. The enzyme known as a-amylase is an important component of saliva and pancreatic juice. (b-Amylase is found in plants. The a- and b-designations for these enzymes serve only to distinguish the two, and do not refer to glycosidic linkage nomenclature.) a-Amylase is an endoglycosidase that hydrolyzes a-(1 ® 4) linkages of amylopectin and glycogen at random positions, eventually producing a mixture of maltose, maltotriose [with three a-(1 ® 4)-linked glucose residues], and other small oligosaccharides. a-Amylase can cleave on either side of a glycogen or amylopectin branch point, but activity is reduced in highly branched regions of the polysaccharide and stops four residues from any branch point.

      The highly branched polysaccharides that are left after extensive exposure to a-amylase are called limit dextrins. These structures can be further degraded by the action of a debranching enzyme, which carries out two distinct reactions. The first of these, known as oligo(a1,4 ® a1,4) glucantransferase activity, removes a trisaccharide unit and transfers this group to the end of another, nearby branch (Figure 23.15). This leaves a single glucose residue in a-(1 ® 6) linkage to the main chain. The a-(1 ® 6) glucosidase activity of the debranching enzyme then cleaves this residue from the chain, leaving a polysaccharide chain with one branch fewer. Repetition of this sequence of events leads to complete degradation of the polysaccharide.

Figure 23.14 · Hydrolysis of glycogen and starch by a-amylase and b-amylase.

      b-Amylase is an exoglycosidase that cleaves maltose units from the free, nonreducing ends of amylopectin branches, as in Figure 23.14. Like a-amylase, however, b-amylase does not cleave either the a-(1 ® 6) bonds at the branch points or the a-(1 ® 4) linkages near the branch points.

Figure 23.15 · The reactions of glycogen debranching enzyme. Transfer of a group of three a-(1 ® 4)-linked glucose residues from a limit branch to another branch is followed by cleavage of the a-(1 ® 6) bond of the residue that remains at the branch point.

Metabolism of Tissue Glycogen

Digestion itself is a highly efficient process in which almost 100% of ingested food is absorbed and metabolized. Digestive breakdown of starch and glycogen is an unregulated process. On the other hand, tissue glycogen represents an important reservoir of potential energy, and it should be no surprise that the reactions involved in its degradation and synthesis are carefully controlled and regulated. Glycogen reserves in liver and muscle tissue are stored in the cytosol as granules exhibiting a molecular weight range from 6x10⁶ to 1600x10⁶. These granular aggregates contain the enzymes required to synthesize and catabolize the glycogen, as well as all the enzymes of glycolysis.
      The principal enzyme of glycogen catabolism is glycogen phosphorylase, a highly regulated enzyme that was discussed extensively in Chapter 15. The glycogen phosphorylase reaction (Figure 23.16) involves phosphorolysis at a nonreducing end of a glycogen polymer. The standard-state free energy change for this reaction is +3.1 kJ/mol, but the intracellular ratio of [P_i] to [glucose-1-P] approaches 100, and thus the actual DG in vivo is approximately -6 kJ/mol. There is an energetic advantage to the cell in this phosphorolysis reaction. If glycogen breakdown were hydrolytic and yielded glucose as a product, it would be necessary to phosphorylate the product glucose (with the expenditure of a molecule of ATP) to initiate its glycolytic degradation.
      The glycogen phosphorylase reaction degrades glycogen to produce limit dextrins, which are further degraded by debranching enzyme, as already described.

Figure 23.16 · The glycogen phosphorylase reaction.

23.4 · Glycogen Synthesis

Animals synthesize and store glycogen when glucose levels are high, but the synthetic pathway is not merely a reversal of the glycogen phosphorylase reaction. High levels of phosphate in the cell favor glycogen breakdown and prevent the phosphorylase reaction from synthesizing glycogen in vivo, in spite of the fact that DG°' for the phosphorylase reaction actually favors glycogen synthesis. Hence, another reaction pathway must be employed in the cell for the net synthesis of glycogen. In essence, this pathway must activate glucose units for transfer to glycogen chains.

Glucose Units Are Activated for Transfer by Formation of Sugar Nucleotides

We are familiar with several examples of chemical activation as a strategy for group transfer reactions. Acetyl-CoA is an activated form of acetate, biotin and tetrahydrofolate activate one-carbon groups for transfer, and ATP is an activated form of phosphate. Luis Leloir,a biochemist in


Figure 23.17 · The structure of UDP-glucose, a sugar nucleotide.

Argentina, showed in the 1950s that glycogen synthesis depended upon sugar nucleotides, which may be thought of as activated forms of sugar units (Figure 23.17). For example, formation of an ester linkage between the C-1 hydroxyl group and the b-phosphate of UDP activates the glucose moiety of UDP-glucose.

UDP-Glucose Synthesis Is Driven by Pyrophosphate Hydrolysis

Sugar nucleotides are formed from sugar-1-phosphates and nucleoside triphosphates by specific pyrophosphorylase enzymes (Figure 23.18). For example, UDP-glucose pyrophosphorylase catalyzes the formation of UDP-glucose from glucose-1-phosphate and uridine 5'-triphosphate:   

              Glucose-1-P + UTP ® UDP-glucose + pyrophosphate

Figure 23.18 · The UDP-glucose pyrophosphorylase reaction is a phosphoanhydride exchange, with a phosphoryl oxygen of glucose-1-P attacking the a-phosphorus of UTP to form UDP-glucose and pyrophosphate.

The reaction proceeds via attack by a phosphate oxygen of glucose-1-phosphate on the a-phosphorus of UTP, with departure of the pyrophosphate anion. The reaction is a reversible one, but—as is the case for many biosynthetic reactions—it is driven forward by subsequent hydrolysis of pyrophosphate:       

                             Pyrophosphate + H₂O ® 2 P_i

The net reaction for sugar nucleotide formation (combining the preceding two equations) is thus    

               Glucose-1-P + UTP + H₂O ® UDP-glucose + 2 P_i

Sugar nucleotides of this type act as donors of sugar units in the biosynthesis of oligo- and polysaccharides. In animals, UDP-glucose is the donor of glucose units for glycogen synthesis, but ADP-glucose is the glucose source for starch synthesis in plants.

Glycogen Synthase Catalyzes Formation of a-(1 ® 4) Glycosidic Bonds in Glycogen

The very large glycogen polymer is built around a tiny protein core. The first glucose residue is covalently joined to the protein glycogenin via an acetal linkage to a tyrosine-OH group on the protein. Sugar units are added to the glycogen polymer by the action of glycogen synthase. The reaction involves transfer of a glucosyl unit from UDP-glucose to the C-4 hydroxyl group at a nonreducing end of a glycogen strand. The mechanism proceeds by cleavage of the C-O bond between the glucose moiety and the Gr-beta-phosphate of UDP-glucose, leaving an oxonium ion intermediate, which is rapidly attacked by the C-4 hydroxyl oxygen of a terminal glucose unit on glycogen (Figure 23.19). The reaction is exergonic and has a DG°' of -13.3 kJ/mol.

Figure 23.19 · The glycogen synthase reaction. Cleavage of the C-O bond of UDP-glucose yields an oxonium intermediate. Attack by the hydroxyl oxygen of the terminal residue of a glycogen molecule completes the reaction.

Glycogen Branching Occurs by Transfer of Terminal Chain Segments

Glycogen is a branched polymer of glucose units. The branches arise from a-(1 ® 6) linkages which occur every 8 to 12 residues. As noted in Chapter 7, the branches provide multiple sites for rapid degradation or elongation of the polymer and also increase its solubility. Glycogen branches are formed by amylo-(1,4  ® 1,6)-transglycosylase, also known as branching enzyme. The reaction involves the transfer of a six- or seven-residue segment from the nonreducing end of a linear chain at least 11 residues in length to the C-6 hydroxyl of a glucose residue of the same chain or another chain (Figure 23.20). For each branching reaction, the resulting polymer has gained a new terminus at which growth can occur.

Figure 23.20 · Formation of glycogen branches by the branching enzyme. Six- or seven-residue segments of a growing glycogen chain are transferred to the C-6 hydroxyl group of a glucose residue on the same or a nearby chain.

23.5 · Control of Glycogen Metabolism

Glycogen Metabolism Is Highly Regulated

Synthesis and degradation of glycogen must be carefully controlled so that this important energy reservoir can properly serve the metabolic needs of the organism. Glucose is the principal metabolic fuel for the brain, and the concentration of glucose in circulating blood must be maintained at about 5 mM for this purpose. Glucose derived from glycogen breakdown is also a primary energy source for muscle contraction. Control of glycogen metabolism is effected via reciprocal regulation of glycogen phosphorylase and glycogen synthase. Thus, activation of glycogen phosphorylase is tightly linked to inhibition of glycogen synthase, and vice versa. Regulation involves both allosteric control and covalent modification, with the latter being under hormonal control. The regulation of glycogen phosphorylase is discussed in detail in Chapter 15.

Regulation of Glycogen Synthase by Covalent Modification

Glycogen synthase also exists in two distinct forms which can be interconverted by the action of specific enzymes: active, dephosphorylated glycogen synthase I (glucose-6-P-independent) and less active phosphorylated glycogen synthase D (glucose-6-P-dependent). The nature of phosphorylation is more complex with glycogen synthase. As many as nine serine residues on the enzyme appear to be subject to phosphorylation, each site’s phosphorylation having some effect on enzyme activity.
                              Dephosphorylation of both glycogen phosphorylase and glycogen synthase is carried out by phosphoprotein phosphatase 1. The action of phosphoprotein phosphatase 1 inactivates glycogen phosphorylase and activates glycogen synthase.

Hormones Regulate Glycogen Synthesis and Degradation

Storage and utilization of tissue glycogen, maintenance of blood glucose concentration, and other aspects of carbohydrate metabolism are meticulously regulated by hormones, including insulin, glucagon, epinephrine, and the glucocorticoids.

Figure 23.21 · The portal vein system carries pancreatic secretions such as insulin and glucagon to the liver and then into the rest of the circulatory system.

The primary hormone responsible for conversion of glucose to glycogen is insulin (Figure 6.36). Insulin is secreted by special cells in the pancreas called the islets of Langerhans. Secretion of insulin is a response to increased glucose in the blood. When blood glucose levels rise (after a meal, for example), insulin is secreted from the pancreas into the pancreatic vein, which empties into the portal vein system (Figure 23.21), so that insulin traverses the liver before it enters the systemic blood supply. Insulin acts to rapidly lower blood glucose concentration in several ways. Insulin stimulates glycogen synthesis and inhibits glycogen breakdown in liver and muscle.
      Several other physiological effects of insulin also serve to lower blood and tissue glucose levels (Figure 23.22). Insulin stimulates the active transport of glucose (and amino acids) across the plasma membranes of muscle and adipose tissue.

Figure 23.22 · The metabolic effects of insulin. As described in Chapter 34, binding of insulin to membrane receptors stimulates the protein kinase activity of the receptor. Subsequent phosphorylation of target proteins modulates the effects indicated.

Insulin also increases cellular utilization of glucose by inducing the synthesis of several important glycolytic enzymes, namely, glucokinase, phosphofructokinase, and pyruvate kinase. In addition, insulin acts to inhibit several enzymes of gluconeogenesis. These various actions enable the organism to respond quickly to increases in blood glucose levels.

A Deeper Look
Carbohydrate Utilization in Exercise
Animals have a remarkable ability to “shift gears” metabolically during periods of strenuous exercise or activity. Metabolic adaptations allow the body to draw on different sources of energy (all of which produce ATP) for different types of activity. During periods of short-term, high-intensity exercise (e.g., a 100-m dash), most of the required energy is supplied directly by existing stores of ATP and creatine phosphate (Figure, part a). Long-term, low-intensity exercise (a 10-km run or a 42.2-km marathon) is fueled almost entirely by aerobic metabolism. Between these extremes is a variety of activities (an 800-m run, for example) that rely on anaerobic glycolysis—conversion of glucose to lactate in the muscles and utilization of the Cori cycle.         For all these activities, breakdown of muscle glycogen provides much of the needed glucose. The rate of glycogen	consumption depends upon the intensity of the exercise (Figure, part b). By contrast, glucose derived from gluconeogenesis makes only small contributions to total glucose consumed during exercise. During prolonged mild exercise, gluconeogenesis accounts for only about 8% of the total glucose consumed. During heavy exercise, this percentage becomes even lower.         Choice of diet has a dramatic effect on glycogen recovery following exhaustive exercise. A diet consisting mainly of protein and fat results in very little recovery of muscle glycogen, even after 5 days (Figure, part c). On the other hand, a high-carbohydrate diet provides faster restoration of muscle glycogen. Even in this case, however, complete recovery of glycogen stores takes about 2 days.
(a) Contributions of the various energy sources to muscle activity during mild exercise. (b) Consumption of glycogen stores in fast-twitch muscles during light, moderate, and heavy exercise. (c) Rate of glycogen replenishment following exhaustive exercise. (a and c adapted from Rhodes and Pflanzer, 1992. Human Physiology. Philadelphia: SaundersCollegePublishing; b adapted from Horton and Terjung, 1988. Exercise, Nutrition and Energy Metabolism. New York : Macmillan.)

Figure 23.23 · The amino acid sequence of glucagon.

Glucagon and Epinephrine Stimulate Glycogen Breakdown

Catabolism of tissue glycogen is triggered by the actions of the hormones epinephrine and glucagon (Figure 23.23). In response todecreased blood glucose, glucagon is released from the a cells in pancreatic islets of Langerhans. This peptide hormone travels through the blood to specific receptors on liver cell membranes. (Glucagon is active in liver and adipose tissue, but not in other tissues.) Similarly, signals from the central nervous system cause release of epinephrine (Figure 23.24)—also known as adrenaline—from the adrenal glands into the bloodstream. Epinephrine acts on liver and muscles. When either hormone binds to its receptor on the outside surface of the cell membrane, a cascade is initiated that activates glycogen phosphorylase and inhibits glycogen synthase. The result of these actions is tightly coordinated stimulation of glycogenbreakdown and inhibition of glycogen synthesis.

Figure 23.24 · Epinephrine

The Phosphorylase Cascade Amplifies the Hormonal Signal

Stimulation of glycogen breakdown involves consumption of molecules of ATP at three different steps in the hormone-sensitive adenylyl cyclase cascade (Figure 15.19). Note that the cascade mechanism is a means of chemical amplification, because the binding of just a few molecules of epinephrine or glucagon results in the synthesis of many molecules of cyclic AMP, which, through the action of cAMP-dependent protein kinase, can activate many more molecules of phosphorylase kinase and even more molecules of phosphorylase. For example, an extracellular level of 10^-10 to 10^-8 M epinephrine prompts the formation of 10^-6 M cyclic AMP, and for each protein kinase activated by cyclic AMP, approximately 30 phosphorylase kinase molecules are activated; these in turn activate some 800 molecules of phosphorylase. Each of these catalyzes the formation of many molecules of glucose-1-P.

The Difference Between Epinephrine and Glucagon

Although both epinephrine and glucagon exert glycogenolytic effects, they do so for quite different reasons. Epinephrine is secreted as a response to anger or fear and may be viewed as an alarm or danger signal for the organism. Called the “fight or flight” hormone, it prepares the organism for mobilization of large amounts of energy. Among the many physiological changes elicited by epinephrine, one is the initiation of the enzyme cascade, as in Figure 15.19, which leads to rapid breakdown of glycogen, inhibition of glycogen synthesis, stimulation of glycolysis, and production of energy. The burst of energy produced is the result of a 2000-fold amplification of the rate of glycolysis. Because a fear or anger response must include generation of energy (in the form of glucose)—both immediately in localized sites (the muscles) and eventually throughout the organism (as supplied by the liver)—epinephrine must be able to activate glycogenolysis in both liver and muscles.
      Glucagon is involved in the long-term maintenance of steady-state levels of glucose in the blood and other tissues. It performs this function by stimulating the liver to release glucose from glycogen stores into the bloodstream. To further elevate glucose levels, glucagon also activates liver gluconeogenesis. It is important to note, however, that stabilization of blood glucose levels is managed almost entirely by the liver. Glucagon does not activate the phosphorylase cascade in muscle (muscle membranes do not contain glucagon receptors). Muscle glycogen breakdown occurs only in response to epinephrine release, and muscle tissue does not participate in maintenance of steady-state glucose levels in the blood.

Cortisol and Glucocorticoid Effects on Glycogen Metabolism

Figure 23.25 · The effects of cortisol on carbohydrate and protein metabolism in the liver.

Glucocorticoids are a class of steroid hormones that exert distinct effects on liver, skeletal muscle, and adipose tissue. The effects of cortisol, a typical glucocorticoid, are best described as catabolic because cortisol promotes protein breakdown and decreases protein synthesis in skeletal muscle. In the liver, however, it stimulates gluconeogenesis and increases glycogen synthesis. Cortisol-induced gluconeogenesis results primarily from increased conversion of amino acids into glucose (Figure 23.25). Specific effects of cortisol in the liver include increased gene expression of several of the enzymes of the gluconeogenic pathway, activation of enzymes involved in amino acid metabolism, and stimulation of the urea cycle, which disposes of nitrogen liberated during amino acid catabolism (Chapter 27).

23.6 · The Pentose Phosphate Pathway

Cells require a constant supply of NADPH for reductive reactions vital to biosynthetic purposes. Much of this requirement is met by a glucose-based metabolic sequence variously called the pentose phosphate pathway, the hexose mono-phosphate shunt, or the phosphogluconate pathway. In addition to providing NADPH for biosynthetic processes, this pathway produces ribose-5-phosphate, which is essential for nucleic acid synthesis. Several metabolites of the pentose phosphate pathway can also be shuttled into glycolysis.

An Overview of the Pathway

The pentose phosphate pathway begins with glucose-6-phosphate, a six-carbon sugar, and produces three-, four-, five-, six-, and seven-carbon sugars (Figure 23.26). As we will see, two successive oxidations lead to the reduction of NADP⁺ to NADPH and the release of CO₂. Five subsequent nonoxidative steps produce a variety of carbohydrates, some of which may enter the glycolytic pathway. The enzymes of the pentose phosphate pathway are particularly abundant in the cytoplasm of liver and adipose cells. These enzymes are largely absent in muscle, where glucose-6-phosphate is utilized primarily for energy production via glycolysis and the TCA cycle. These pentose phosphate pathway enzymes are located in the cytosol, which is the site of fatty acid synthesis, a pathway heavily dependent on NADPH for reductive reactions.Figure 23.26 · The pentose phosphate pathway. The numerals in the blue circles indicate the steps discussed in the text.

The Oxidative Steps of the Pentose Phosphate Pathway

(1) Glucose-6-Phosphate Dehydrogenase

Figure 23.27 · The glucose-6-phosphate dehydrogenase reaction is the committed step in the pentose phosphate pathway.

The pentose phosphate pathway begins with the oxidation of glucose-6-phosphate. The products of the reaction are a cyclic ester (the lactone of phosphogluconic acid) and NADPH (Figure 23.27). Glucose-6-phosphate dehydrogenase, which catalyzes this reaction, is highly specific for NADP⁺. As the first step of a major pathway, the reaction is irreversible and highly regulated. Glucose-6-phosphate dehydrogenase is strongly inhibited by the product coenzyme, NADPH, and also by fatty acid esters of coenzyme A (which are intermediates of fatty acid biosynthesis). Inhibition due to NADPH depends upon the cytosolic NADP⁺/NADPH ratio, which in the liver is about 0.015 (compared to about 725 for the NAD⁺/NADH ratio in the cytosol).

(2) Gluconolactonase

Figure 23.28 · The gluconolactonase reaction.

The gluconolactone produced in step 1 is hydrolytically unstable and readily undergoes a spontaneous ring-opening hydrolysis, although an enzyme, gluconolactonase, accelerates this reaction (Figure 23.28). The linear product, the sugar acid 6-phospho-D-gluconate, is further oxidized in step 3.

(3) 6-Phosphogluconate Dehydrogenase

Figure 23.29 · The 6-phosphogluconate dehydrogenase reaction.

The oxidative decarboxylation of 6-phosphogluconate by 6-phosphogluconate dehydrogenase yields D-ribulose-5-phosphate and another equivalent of NADPH. There are two distinct steps in this reaction (Figure 23.29): the initial NADP⁺-dependent dehydrogenation yields a b-keto acid, 3-keto-6-phosphogluconate, which is very susceptible to decarboxylation (the second step). The resulting product, D-ribulose-5-P, is the substrate for the nonoxidative reactions composing the rest of this pathway.

The Nonoxidative Steps of the Pentose Phosphate Pathway

This portion of the pathway begins with an isomerization and an epimerization, and it leads to the formation of either D-ribose-5-phosphate or D-xylulose-5-phosphate. These intermediates can then be converted into glycolytic intermediates or directed to biosynthetic processes.

(4) Phosphopentose Isomerase

This enzyme interconverts ribulose-5-P and ribose-5-P via an enediol intermediate (Figure 23.30). The reaction (and mechanism) is quite similar to the phosphoglucoisomerase reaction of glycolysis, which interconverts glucose-6-P and fructose-6-P. The ribose-5-P produced in this reaction is utilized in the biosynthesis of coenzymes (including NADH, NADPH, FAD, and B₁₂), nucleotides, and nucleic acids (DNA and RNA). The net reaction for the first four steps of the pentose phosphate pathway is

            Glucose-6-P+2 NADP⁺ ® ribose-5-P+2 NADPH+2 H⁺+CO₂

Figure 23.30 · The phosphopentose isomerase reaction involves an enediol intermediate.

(5) Phosphopentose Epimerase

This reaction converts ribulose-5-P to another ketose, namely, xylulose-5-P. This reaction also proceeds by an enediol intermediate, but involves an inversion at C-3 (Figure 23.31). In the reaction, an acidic proton located a- to a carbonyl carbon is removed to generate the enediolate, but the proton is added back to the same carbon from the opposite side. Note the distinction in nomenclature here. Interchange of groups on a single carbon is an epimerization, and interchange of groups between carbons is referred to as an isomerization.

Figure 23.31 · The phosphopentose epimerase reaction interconverts ribulose-5-P and xylulose-5-phosphate. The mechanism involves an enediol intermediate and occurs with inversion at C-3.

      To this point, the pathway has generated a pool of pentose phosphates. The DG°' for each of the last two reactions is small, and the three pentose-5-phosphates coexist at equilibrium. The pathway has also produced two molecules of NADPH for each glucose-6-P converted to pentose-5-phosphate. The next three steps rearrange the five-carbon skeletons of the pentoses to produce three-, four-, six-, and seven-carbon units, which can be used for various metabolic purposes. Why should the cell do this? Very often, the cellular need for NADPH is considerably greater than the need for ribose-5-phosphate. The next three steps thus return some of the five-carbon units to glyceraldehyde-3-phosphate and fructose-6-phosphate, which can enter the glycolytic pathway. The advantage of this is that the cell has met its needs for NADPH and ribose-5-phosphate in a single pathway, yet at the same time it can return the excess carbon metabolites to glycolysis.

(6) and (8) Transketolase

Figure 23.32 · The transketolase reaction of step 6 in the pentose phosphate pathway.

The transketolase enzyme acts at both steps 6 and 8 of the pentose phosphate pathway. In both cases, the enzyme catalyzes the transfer of two-carbon units. In these reactions (and also in step 7, the transaldolase reaction, which transfers three-carbon units), the donor molecule is a ketose and the recipient is an aldose. In step 6, xylulose-5-phosphate transfers a two-carbon unit to ribose-5-phosphate to form glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate (Figure 23.32). Step 8 involves a two-carbon transfer from xylulose-5-phosphate to erythrose 4-phosphate to produce

Figure 23.33 · The transketolase reaction of step 8 in the pentose phosphate pathway.

another glyceraldehyde-3-phosphate and a fructose-6-phosphate (Figure 23.33).Three of these products enter directly into the glycolytic pathway. (The sedoheptulose-7-phosphate is taken care of in step 7, as we shall see.) Transketolase is a thiamine pyrophosphate-dependent enzyme, and the mechanism (Figure 23.34) involves abstraction of the acidic thiazole proton of TPP, attack by the resulting carbanion at the carbonyl carbon of the ketose phosphate substrate, expulsion of the glyceraldehyde-3-phosphate product, and transfer of the two-carbon unit. Transketolase can process a variety of 2-keto sugar phosphates in a similar manner. It is specific for ketose substrates with the configuration shown, but can accept a variety of aldose phosphate substrates.

Figure 23.34 · The mechanism of the TPP-dependent transketolase reaction. Ironically, the group transferred in the transketolase reaction might best be described as an aldol, whereas the transferred group in the transaldolase reaction is actually a ketol. Despite the irony, these names persist for historical reasons.

(7) Transaldolase

The transaldolase functions primarily to make a useful glycolytic substrate from the sedoheptulose-7-phosphate produced by the first transketolase reaction. This reaction (Figure 23.35) is quite similar to the aldolase reaction of glycolysis, involving formation of a Schiff base intermediate between the sedoheptulose-7-phosphate and an active-site lysine residue (Figure 23.36). Elimination of the erythrose-4-phosphate product leaves an enamine of dihydroxyacetone, which remains stable at the active site (without imine ydrolysis) until the other substrate comes into position. Attack of the enamine carbanion at the carbonyl carbon of glyceraldehyde-3-phosphate is followed by hydrolysis of the Schiff base (imine) to yield the product fructose-6-phosphate.

Figure 23.35 · The transaldolase reaction.

Figure 23.36 · The transaldolase mechanism involves attack on the substrate by an active-site lysine. Departure of erythrose-4-P leaves the reactive enamine, which attacks the aldehyde carbon of glyceraldehyde-3-P. Schiff base hydrolysis yields the second product, fructose-6-P.

Utilization of Glucose-6-P Depends on the Cell’s Need for ATP, NADPH, and Ribose-5-P

It is clear that glucose-6-phosphate can be used as a substrate either for glycolysis or for the pentose phosphate pathway. The cell makes this choice on the basis of its relative needs for biosynthesis and for energy from metabolism. ATP can be produced in abundance if glucose-6-phosphate is channeled into glycolysis. On the other hand, if NADPH or ribose-5-phosphate is needed, glucose-6-phosphate can be directed to the pentose phosphate pathway. The molecular basis for this regulatory decision depends on the enzymes that metabolize glucose-6-phosphate in glycolysis and the pentose phosphate pathway. In glycolysis, phosphoglucoisomerase converts glucose-6-phosphate to fructose-6-phosphate, which is utilized by phosphofructokinase (a highly regulated enzyme) to produce fructose-1,6-bisphosphate. In the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (also highly regulated) produces gluconolactone from glucose-6-phosphate. Thus, the fate of glucose-6-phosphate is determined to a large extent by the relative activities of phosphofructokinase and glucose-6-P dehydrogenase. Recall (Chapter 19) that PFK is inhibited when the ATP/AMP ratio increases, and that it is inhibited by citrate but activated by fructose-2,6-bisphosphate. Thus, when the energy charge is high, glycolytic flux decreases. Glucose-6-P dehydrogenase, on the other hand, is inhibited by high levels of NADPH and also by the intermediates of fatty acid biosynthesis. Both of these are indicators that biosynthetic demands have been satisfied. If that is the case, glucose-6-phosphate dehydrogenase and the pentose phosphate pathway are inhibited. If NADPH levels drop, the pentose phosphate pathway turns on, and NADPH and ribose-5-phosphate are made for biosynthetic purposes.
      Even when the latter choice has been made, however, the cell must still be “cognizant” of the relative needs for ribose-5-phosphate and NADPH (as well as ATP). Depending on these relative needs, the reactions of glycolysis and the pentose phosphate pathway can be combined in novel ways to emphasize the synthesis of needed metabolites. There are four principal possibilities.

(1) BOTH RIBOSE-5-P AND NADPH ARE NEEDED BY THE CELL      In this case, the first four reactions of the pentosephosphate pathway predominate (Figure 23.37). NADPH is produced by the oxidative reactions of the pathway, and ribose-5-P is the principal product of carbon metabolism. As stated earlier, the net reaction for these processes is

Figure 23.37 · When biosynthetic demands dictate, the first four reactions of the pentose phosphate pathway predominate and the principal products are ribose-5-P and NADPH.

 Glucose-6-P + 2 NADP⁺ + H₂O ® ribose-5-P + CO₂ + 2 NADPH + 2 H⁺

(2) MORE RIBOSE-5-P THAN NADPH IS NEEDED BY THE CELL      Synthesis of ribose-5-P can be accomplished without production of NADPH if the oxidative steps of the pentose phosphate pathway are bypassed. The key to this route is the extraction of fructose-6-P and glyceraldehyde-3-P, but not glucose-6-P, from glycolysis (Figure 23.38). The action of transketolase and transaldolase on fructose-6-P and glyceraldehyde-3-P produces three molecules of ribose-5-P from two mole-cules of fructose-6-P and one of glyceraldehyde-3-P. In this route, as in case 1, no carbon metabolites are returned to glycolysis. The net reaction for this route is

                  5 Glucose-6-P + ATP ® 6 ribose-5-P + ADP + H⁺

Figure 23.38 · The oxidative steps of the pentose phosphate pathway can be bypassed if the primary need is for ribose-5-P.

(3) MORE NADPH THAN RIBOSE-5-P IS NEEDED BY THE CELL      Large amounts of NADPH can be supplied for biosynthesis without concomitant production of ribose-5-P, if ribose-5-P produced in the pentose phosphate pathway is recycled to produce glycolytic intermediates. As shown in Figure 23.39, this alternative involves a complex interplay between the transketolase and transaldolase reactions to convert ribulose-5-P to fructose-6-P and glyceraldehyde-3-P, which can be recycled to glucose-6-P via gluconeogenesis. The net reaction for this process is

6 Glucose-6-P + 12 NADP⁺ + 6 H₂O ®
                          6 ribulose-5-P + 6 CO₂ + 12 NADPH + 12 H⁺
                                 6 Ribulose-5-P ® 5-glucose-6-P + P_i

Figure 23.39 · Large amounts of NADPH can be produced by the pentose phosphate pathway without significant net production of ribose-5-P. In this version of the pathway, ribose-5-P is recycled to produce glycolytic intermediates.

Note that in this scheme, the six hexose sugars have been converted to six pentose sugars with release of six molecules of CO₂, and the six pentoses are reconverted to five glucose molecules.

(4) BOTH NADPH AND ATP ARE NEEDED BY THE CELL, BUT RIBOSE-5-P IS NOT      Under some conditions, both NADPH and ATP must be provided in the cell. This can be accomplished in a series of reactions similar to case 3, if the fructose-6-P and glyceraldehyde-3-P produced in this way proceed through glycolysis to produce ATP and pyruvate, which itself can yield even more ATP by continuing on to the TCA cycle (Figure 23.40). The net reaction for this alternative is

3 Glucose-6-P + 5 NAD⁺ + 6 NADP⁺ + 8 ADP + 5 P_i ®
          5 pyruvate + 3 CO₂ + 5 NADH + 6 NADPH + 8 ATP + 2 H₂O + 8 H⁺

Figure 23.40 · Both ATP and NADPH (as well as NADH) can be produced by this version of the pentose phosphate and glycolytic pathways.

Note that, except for the three molecules of CO₂, all the other carbon from glucose-6-P is recovered in pyruvate.