Bioc 460 - Dr.

Miesfeld Fall 2008

Lecture 32 - Carbohydrate Metabolism Key Concepts - Pentose Phosphate Pathway Enzymatic reactions in the oxidative phase Enzymatic reactions in the nonoxidative phase Glucose-6P dehydrogenase deficiency in humans - Gluconeogenesis Three steps in glycolysis are bypassed by gluconeogenesis Reciprocal control of glycolysis and gluconeogenesis The Cori Cycle provides glucose to muscle cells during exercise Key Questions about the Pentose Phosphate Pathway and Gluconeogenesis: What is the biochemical basis for favism and how is it related to malarial resistance? How does fructose-2,6-bisphosphate control flux through gluconeogenesis and glycolysis? Biochemical Applications: Monitoring blood glucose levels throughout the day is critical to diabetics who need insulin injections. Glucose monitoring devices are based on an assay using the enzyme glucose oxidase which produces gluconate and hydrogen peroxide (H2O2) from glucose. The level of H2O2 in the sample is detected by an indicator dye that is oxidized in a reaction catalyzed by peroxidase. In lectures 22-32, we examined the regulation of energy conversion pathways in cells and saw how the absorption of sunlight by photosynthetic organisms was used as an energy source to drive carbohydrate synthesis in the form of starch and sucrose. We now begin the second half of our journey through the metabolic forest by exploring biochemical pathways in the cell that control the synthesis and degradation of a variety of biomolecules. The energy required for biomolecular synthesis comes from phosphoryl transfer energy available from ATP, and from a redox energy provided by the phosphorylated form of NADH called NADPH. Degradation of biomolecules is an important process in cells because it not only scavenges Figure 1. building blocks for biosynthesis, but it is a form of metabolic regulation by controlling the steady-state level of active biomolecules. The three primary pathways in anabolic carbohydrate metabolism in non-photosynthetic organisms are the pentose phosphate pathway, gluconeogenesis and glycogen metabolism. We describe the pentose phosphate pathway and gluconeogenesis here in lecture 33, and then discuss glycogen metabolism in lectures 34 (enzyme reactions) and 35 (regulation of glycogen metabolism). The major sources of carbon in gluconeogenesis are amino acids and glycerol in animals, and glyceraldehyde-3-phosphate (GAP) in plants. Metabolism of ribose sugars in the pentose phosphate pathway is used to generate NADPH and to provide the carbohydrate component of nucleotides (figure 1).
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Pentose Phosphate Pathway The pentose phosphate pathway takes place entirely within the cytoplasm and is also known as the hexose monophosphate shunt or phosphogluconate pathway. The most important function of the pentose phosphate pathway is to reduce two molecules of NADP+ to NADPH for each glucose6-phosphate (glucose-6P) that is oxidatively decarboxylated to ribulose-5-phosphate (ribulose-5P). The pentose phosphate pathway is also responsible for producing ribose-5-phosphate (ribose5P) from glucose-6P. NADPH (nicotinamide adenine dinucleotide phosphate) is structurally identical to NADH with the exception of a phosphate group on the C-2 carbon of the sugar moiety of the adenine nucleotide. Another difference between the conjugate redox pairs of NAD+/NADH and NADP+/NADPH is that NAD+ functions as the primary oxidant in the cell (accepts electrons), whereas, NADPH is the primary reductant in the cell (donates electrons). The distinct roles of these two related coenzymes can be seen in the very different steady-state levels of the conjugate redox pairs. In liver cells, the [NAD+]/[NADH] ratio is close to 1,000, whereas, the [NADP+]/[NADPH] ratio is 0.01, i.e., NAD+ and NADPH are the most abundant redox species. The pentose phosphate pathway can be divided into two phases, the oxidative phase which generates NADPH, and the nonoxidative phase, which interconverts C3, C4, C5, C6 and C7 sugar phosphates using many of the same "carbon shuffle" reactions we saw in the Calvin cycle. Figure 2 provides an overview of the pentose Figure 2. phosphate pathway illustrating the function of the oxidative and nonoxidative phases in producing NADPH and ribose-5P, respectively. Flux through the oxidative and nonoxidative phases of the pathway is tightly regulated in response to energy needs of the cell, the NADP+/NADPH ratio, and requirements for nucleotide and coenzyme biosynthesis (see #1, #2 and #3 in figure 2). For example, when NADPH is needed, ribulose-5P is converted back into glucose-6P to maintain flux through the pathway (#1), however, if ATP and NADPH are needed (which would be the case for most anabolic pathways), then some of the ribulose-5P is used to synthesis hexose phosphates for glycolysis (#2). Finally, if the cell needs to increase the rate of nucleotide and coenzyme biosynthesis, then most of the ribulose5P is shunted toward ribose-5P synthesis (#3). 1. What does the pentose phosphate pathway accomplish for the cell? The oxidative phase generates NADPH which is required for many biosynthetic pathways and for detoxification of reactive oxygen species. The nonoxidative phase interconverts C3, C4, C5, C6 and C7 monosaccharides to produce ribose5P for nucleotide synthesis, and also to regenerate glucose-6P to maintain NADPH production by the oxidative phase. 2. What is the overall net reaction of the pentose phosphate pathway when it is utilized to generate the maximum amount of NADPH? 6 Glucose-6P + 12 NADP+ + 12 H2O --> 5 Glucose-6P + 12 NADPH + 12 H+ + 6 CO2
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3. What are the key enzymes in the pentose phosphate pathway? Glucose-6P dehydrogenase (G6PD) enzyme catalyzing the first reaction in the pathway which converts glucose-6P to 6-phosphogluconolactone. This reaction is the commitment step in the pathway and is feedback-inhibited by NADPH. Defects in glucose-6P dehydrogenase cause a dietary condition called favism. Transketolase and Transaldolase - together these two enzyme catalyze the reversible "carbon shuffle" reactions of the nonoxidative phase of the pathway. Transketolase catalyzes the transfer of C2 units between sugars and transaldolase catalyzes the transfer of C3 units. These are the same enzymes used in the Calvin cycle to regenerate ribulose-5P from glyceraldehyde-3P. 4. What are examples of the pentose phosphate pathway in real life? Glucose-6P dehydrogenase deficiency is the most common enzyme deficiency in the world and affects over 400 million people. A 90% decrease in enzyme activity results in the inability of red blood cells to produce enough NADPH to protect the cells from reactive oxygen species that are generated by anti-malarial drugs and by compounds in fava beans. Enzymatic reactions in the oxidative phase As shown in figure 3, the oxidative phase of the pentose phosphate pathway includes three enzymatic reactions, the first of which is catalyzed by the enzyme glucose-6P dehydrogenase (G6PD). This irreversible reaction (G' = -17.6 kJ/mol) represents the commitment step in the pathway because the product, 6-phosphgluconolactone has no other metabolic fates. The oxidation of Figure 3. glucose-6P by glucose-6P dehydrogenase is coupled to the reduction of NADP+ resulting in the formation of one molecule of NADPH. In the second reaction, 6-phosphogluconolactone is hydrolyzed by lactonase to produce the open chain monosaccharide 6-phosphogluconate. Finally, 6-phosphogluconate is oxidized and decarboxylated in a reaction catalyzed by Figure 4. 6-phosphogluconate dehydrogenase to generate ribulose-5P, CO2 and the second molecule of NADPH. Enzymatic reactions in the nonoxidative phase In cells that require high levels of NADPH for biosynthetic reactions, the ribulose-5P produced in the oxidative phase needs to be converted back into glucose-6P to maintain flux through the glucose-6P dehydrogenase reaction. Figure 4 shows the carbon shuffle reactions of the
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nonoxidative phase which ultimately are used to regenerate glucose-6P using the same transketolase and transaldolase enzyme reactions that we saw in the Calvin cycle (lecture 32). Since enzymes in the pentose phosphate pathway are localized to the cytosol, as are the enzymes for glycolysis and gluconeogenesis, fructose-6P is readily converted to glucose-6P by the enzyme phosphoglucose isomerase. Similarly, glyceraldehyde-3P is converted to fructose-6P by a series of three reactions involving the enzymes Figure 5. triosephosphate isomerase, aldolase and fructose-1,6-bisphosphatase. With the exception of fructose-1,6-bisphosphatase which catalyzes a highly exergonic reaction in gluconeogenesis (G'=-16.3 kJ/mol), the other three enzymes function at or near equilibrium in both the glycolytic and gluconeogenic pathways. Figure 5 illustrates how six C5 molecules (ribose-5P and xylulose-5P) are used to resynthesize five C6 molecules (glucose-6P) in the nonoxidative phase. Since glucose-6P is a substrate for both the glycolytic pathway and the pentose phosphate pathway, what controls flux through these two pathways? As shown in figure 6, when the rates of NADPH-dependent biosynthetic reactions are high in the cytosol, then the [NADP+]/[NADPH] ratio increases, leading to allosteric activation of glucose-6P dehydrogenase activity by NADP+. This in turn, increases flux through the pentose phosphate pathway to produce more NADPH by stimulating oxidative decarboxylation of glucose-6P by enzymes in the oxidative phase of the pathway. When the level of NADPH rises in the cell, it competes with NADP+ for binding to glucose-6P dehydrogenase, thereby Figure 6. reducing the activity of the enzyme. This results in decreased flux through the pentose phosphate pathway and the available glucose-6P is then metabolized by the glycolytic pathway as a source of energy for the production of ATP. This makes sense because biosynthetic pathways require ATP, and when ATP levels drop (low energy charge in the cell), the demand for NADPH will also diminish causing glucose6P to be shunted away from the pentose phosphate pathway and toward glycolysis. Glucose-6P dehydrogenase deficiency in humans In addition to its role in generating NADPH for biosynthetic pathways in the liver (primarily fatty acid and cholesterol biosynthesis), the pentose phosphate pathway is also responsible for maintaining high levels of NADPH in red blood cells (erythrocytes) for use as a reductant in the glutathione reductase reaction shown in figure 7. Glutathione is a tripeptide (glutamylcysteinylglycine) that has a free sulfhydryl group which functions as an electron donor in a variety of coupled redox reactions in the cell. In erythrocytes, electrons from glutathione are used to keep cysteine residues in hemoglobin in the reduced state, and for reducing harmful reactive oxygen species and hydroxyl free radicals that damage proteins and lipids through oxidationinduced cleavage reactions. Glutathione reductase is a flavoprotein that contains the coenzyme
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FAD and is related to ferredoxin-NADP+ reductase. Figure 7. As shown in figure 8, glutathione reductase uses the two electrons available from NADPH to maintain glutathione in the reduced state (GSSG ---> 2 GSH). High levels of GSH, and therefore high levels of NADPH, are needed in erythrocytes to reduce hydrogen peroxide (H2O2) levels through a GSHdependent redox reaction catalyzed by the enzyme glutathione peroxidase. When erythrocytes are exposed to chemicals that generate high levels of superoxide radicals, GSH is required to reduce these damaging compounds. An active pentose phosphate pathway in erythrocytes normally provides sufficient levels of NADPH to maintain the GSH:GSSG ratio at about 500:1. Glucose-6P dehydrogenase (G6PD) deficiency is the most common enzyme deficiency in the world, effecting over 400 million people. The discovery of G6PD deficiency in Figure 8. the mid 1950s came as a result of observations made 30 years earlier when it was noticed that the anti-malarial drug primaquine induced acute hemolytic anemia (red blood cell lysis) in a small percentage of people who had been given primaquine prophylatically. The biochemical basis for this drug-induced illness was found to be lower than normal levels of NADPH in erythrocytes due to a G6PD deficiency. People with G6PD deficiency cannot tolerate primaquine because their erythrocytes do not contain enough GSH to detoxify the reactive oxygen species produced by the compound. In fact, the reason primaquine works as an anti-malarial drug is because productive infection of the mosquito-borne microorganism Plasmodium is inhibited in erythrocytes under conditions in which NADPH levels are reduced due to increased oxidative stress. Figure 9 shows the chemical structure of anti-malarial drug primaquine, as Figure 9. well as, vicine, a compound found at high levels in fava beans. Interestingly, it has been found that people who inherit the G6PD mutation actually have lower incidence of malarial infection. The explanation for this is that reduced levels of NADPH, and the associated increase in oxidative stress in erythrocytes (coming from normal biochemical processes in the cell), creates a hostile environment for the malarial pathogen. This would be analogous to how the hemoglobin S gene defect (HbS), which causes sickle cell anemia, affords protection against malaria because of the reduced ability of the pathogen to infect HbS-containing cells. The finding that people with G6PD deficiency are for the most part asymptomatic (no signs of illness), but can get gravely sick when given primaquine, led to the realization that another
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mysterious illness called favism was also caused by the same enzyme defect. As far back as the 6th century B.C. in the times of Pythagoras, it was observed that if certain people ate foods containing fava beans, a main ingredient in the Mediterranean dish falafel, they would become very sick. It is now known that the same acute hemolytic anemia seen in individuals with G6PD who are treated with primaquine (or any other drug that induces oxidative stress in erythrocytes), also explains the symptoms of favism. One of the active compounds in fava beans is called vicine, a toxic glycoside that induces oxidative stress in erythrocytes. Gluconeogenesis Glucose is the primary chemical energy source for most nonphotosynthetic organisms (and for plants at night), and therefore must be readily available at all times. When dietary sources of glucose are insufficient, and glucose stores have been depleted (starch in plants and glycogen in animals), glucose is synthesized from non-carbohydrate compounds by a series of cytosolic reactions called the gluconeogenic pathway as shown in figure 10. Gluconeogenesis converts pyruvate to glucose using a set of reactions that require energy input in the form of ATP and GTP (gluconeogenesis costs 4 ATP and 2 GTP to synthesize one glucose from two pyruvate). Importantly, gluconeogenesis is not simply the reversal of glycolysis, and is in fact, a highly regulated pathway (as is glycolysis) to prevent futile cycling between glucose degradation by glycolysis and glucose synthesis by gluconeogenesis.
Figure 10.

1. What does gluconeogenesis accomplish for the organism? The liver and kidney generate glucose from noncarbohydrate sources (lactate, amino acids, glycerol) for export to other tissues that depend on glucose for energy, primarily the brain and erythrocytes. Plants use the gluconeogenic pathway to convert GAP, the product of the Calvin cycle, into glucose which is used to make sucrose and starch. 2. What is the overall net reaction of gluconeogenesis? 2 pyruvate + 2NADH + 4ATP + 2GTP + 6H2O Glucose + 2NAD+ + 2H+ + 4ADP + 2GDP + 6Pi 3. What are the key enzymes in gluconeogenesis? Pyruvate carboxylase is a mitochondrial enzyme that catalyzes a carboxylation reaction converting pyruvate to oxaloacetate using a reaction mechanism involving a biotinyl "swinging arm" and ATP hydrolysis. Pyruvate carboxylase is dependent on allosteric activation by acetyl CoA. Phosphoenolpyruvate carboxykinase (PEPCK) is localized to either the mitochondrial matrix or the cytosol (or both in the case of human liver cells) and catalyzes a phosphoryl transfer reaction that converts oxaloacetate to phosphoenolpyruvate (PEP) using the energy released by decarboxylation and GTP hydrolysis. Transcription of the PEPCK gene is regulated by hormones.

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Fructose-1,6-bisphosphatase-1 (FBPase-1) - catalyzes the dephosphorylation of fructose-1,6BP to form fructose-6P; this is the bypass reaction for PFK-1 in glycolysis. FBPase-1 is inhibited by the allosteric regulators F2,6BP and AMP Figure 11. which are also allosteric activators of PFK-1. Glucose-6-phosphatase - is an enzyme in liver and kidney cells (not present in muscle cells) that catalyzes the dephosphorylation of glucose-6P to form glucose which can be exported out of the cell. Glucose-6phosphatase is located in the lumen of the endoplasmic reticulum. 4. What are examples of gluconeogenesis in real life? Athletes that exercise intensely for short periods of time, such as in a sprint race, build up large amounts of lactate in their muscles as a result of anaerobic glycolysis. The "warming down" period of continual movement under aerobic conditions performed by athletes for ~15 minutes after a race increases circulation and removes lactate from the muscle. The lactate is transported to the liver where it is converted to glucose by the gluconeogenic pathway and shipped back to the muscle to replenish glycogen. This process is called the Cori cycle. Three steps in glycolysis are bypassed by gluconeogenesis As shown in figure 11, glycolysis and gluconeogenesis are opposing pathways that serve the critical function of degrading or synthesizing glucose in response to energy demands in the cell (glycolysis to generate ATP), and in the whole animal (gluconeogenesis to export glucose). These two pathways share seven of the same enzymes, Figure 12. with additional pathway-specific enzymes required at the three key regulatory steps shown in figure 11. Two of the bypass enzymes in gluconeogenesis, fructose-1,6bisphosphatase-1 (FBPase-1) and glucose-6phosphatase, simply reverse the reaction catalyzed by the corresponding glycolytic enzymes phosphofructokinase-1 (PFK-1) and hexokinase, respectively. However, as shown in figure 12, two gluconeogenic enzymes, pyruvate carboxylase and phosphoenolypyruvate carboxykinase (PEPCK), are required to catalyze the bypass reaction that converts pyruvate to PEP. Pyruvate carboxylase is a mitochondrial enzyme that requires the cofactor biotin to function as a carboxyl group carrier in a two step enzyme reaction. Pyruvate carboxylase is activated by acetyl CoA
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and has an important role in supplying OAA to the citrate cycle when acetyl CoA levels are high and the energy charge in the cell is low. The cellular location of PEPCK differs depending on the species. Humans actually contain two distinct PEPCK genes that encode mitochondrial and cytosolic PEPCK enzymes. As shown in figure 13, when NADH equivalents need to be moved from the mitochondrial matrix Figure 13. to the cytosol to maintain flux through the glyceraldehyde3P dehydrogenase reaction in gluconeogenesis (e.g., when amino acids are used as a source of pyruvate), the oxaloacetate generated by pyruvate carboxylase in the mitochondria is converted to malate which is shuttled to the cytosol. Oxidation of this malate by cytosolic malate dehydrogenase results in the generation of NADH in the cytosol, thereby effectively transporting NADH equivalents from the mitochondrial matrix to the cytosol. The cytosolic oxaloacetate is then converted to PEP by cytosolic PEPCK. Figure 13 also shows an alternate pathway from pyruvate to PEP that is utilized in humans when lactate builds up due to anaerobic metabolism in muscle cells (lactate is transported to the liver as part of the Cori cycle). In this case, oxidation of lactate by lactate dehydrogenase in the cytosol generates pyruvate and the necessary NADH for the glyceraldehyde-3P dehydrogenase reaction without requiring the malate shuttle. The oxaloacetate produced in the mitochondria by pyruvate carboxylase is converted to PEP by mitochondrial PEPCK. The PEP produced in the mitochondria is shuttled by a specific transport system to the cytosol where it enters gluconeogenesis. Reciprocal control of glycolysis and gluconeogenesis As shown in figure 14, the activities of PFK-1 and FBPase-1 are regulated by the allosteric effectors AMP, citrate and fructose-2,6-bisphosphate (F-2,6-BP), but in a reciprocal manner. Reciprocal regulation refers to the fact that the same regulatory molecule has opposite effects on two enzymes Figure 14. that control a shared step in two reaction pathways. For example, when energy charge in the cell is low, AMP levels are high leading to activation of PFK-1 (increased flux through glycolysis) and inhibition of FBPase-1 (decreased flux through gluconeogenesis). This makes sense because the pyruvate generated by glycolysis can then be used in the energy conversion pathways to replenish ATP, while at the same time, glucose synthesis is shutdown resulting in a
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build-up of pyruvate. In contrast, when citrate levels are high in the cytosol it means that the citrate cycle is backed up and glycolysis needs to be inhibited while at the same time converting the leftover pyruvate to glucose by activating gluconeogenesis. The allosteric regulator F-2,6-BP is an even more potent regulator of these two enzymes than either AMP or citrate. Note that while F2,6-BP is structurally related to fructose-6P and fructose-1,6BP (the only difference is the position of the phosphate groups), F-2,6-BP not a metabolic intermediate in either the glycolytic or gluconeogenic pathways, instead it is an allosteric regulator that activates PFK-1 and inhibits FBPase-1. As shown in figure 15, in the Figure 15. presence of F-2,6-BP, the affinity of PFK1 for its substrate fructose-6P is 25 times higher than it is in the absence of F2,6BP. Looking at the activity curves for FBPase-1 in the presence and absence of F-2,6-BP it can be seen that the affinity of FBPase-1 for its substrate fructose1,6BP is 15 times lower in the presence of F-2,6-BP. As with other allosteric regulators, F-2,6-BP binds to PFK-1 and FBPase-1 at sites outside of the active site resulting in protein conformational changes that effect substrate binding affinities. The amount of F-2,6-BP in the cell is regulated by hormone signaling through glucagon and insulin which control the activity of a dual function enzyme containing two catalytic activities, 1) a kinase activity called phosphofructokinase-2 (PFK-2) that Figure 16. phosphorylates fructose-6P to form F-2,6-BP, and 2) a phosphatase activity called fructose-2,6-bisphosphatase (FBPase-2) that dephosphorylates F-2,6-BP to form fructose6P (figure 16). When the PFK-2/FBPase-2 dual function enzyme is unphosphorylated, then the PFK-2 activity in the enzyme is stimulated and the FBPase-2 activity is inhibited, resulting in the net phosphorylation of fructose-6P to produce more F-2,6-BP which stimulates glycolytic flux. In contrast, when PFK-2/FBPase-2 is phosphorylated, the activity of PFK-2 is inhibited and the activity of FBPase-2 is stimulated. Under these conditions, the PFK-2/FBPase-2 enzyme dephosphorylates F-2,6-BP resulting in lower levels of F-2,6-BP which Figure 17. stimulates gluconeogenic flux by "derepressing" FBPase-1 activity. Figure 17 shows that activation of the glucagon receptor in liver cells results in stimulation of
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protein kinase A signaling which leads to phosphorylation of the PFK-2/FBPase-2 enzyme, thereby leading to decreased levels of F-2,6-BP and increased activity of the gluconeogenic enzyme FBPase-1. In contrast, insulin signaling stimulates protein phosphatase-1 activity resulting in the dephosphorylation of the PFK-2/FBPase-2 enzyme leading to higher levels of F2,6-BP and activation of the glycolytic enzyme PFK-1. Taken together, glucagon signaling stimulates gluconeogenesis to elevate blood glucose levels, while insulin stimulates glycolysis which serves to lower blood glucose levels by increasing the rate of glucose degradation inside the cell. The Cori Cycle provides glucose to muscle cells during exercise Regulating metabolic flux through glycolysis and gluconeogenesis in the same cell requires that the opposing enzymes in both pathways be reciprocally regulated to avoid futile cycling (ATP hydrolysis in the absence of net chemical work). Figure 18. However, there is one situation in which having both pathways active at the same time, but in different cells, can be quite advantageous. As shown in figure 18, the Cori cycle, which was first described by Carl and Gerty Cori in 1929, provides a mechanism to convert lactate produced by anaerobic glycolysis in muscle cells to glucose using the gluconeogenic pathway in liver cells. Although it costs four high energy phosphate bonds to run the Cori cycle (the difference between 2 ATP produced by anaerobic glycolysis and 4 ATP and 2 GTP consumed by gluconeogenesis), the benefit to the organism is that glycogen stores in the muscle can be quickly replenished following prolonged exercise. Studies on athletes have shown that within 30 minutes of completing a vigorous workout, the majority of lactate produced during anaerobic glycolysis in the muscle has been converted to glucose in the liver and used to replenish muscle glycogen stores. In fact, the reason you should "warm down" after exercise (same movement but under aerobic conditions) is to enhance circulation so that lactate will be cleared from the muscle and be used in the liver for glucose synthesis via the Cori cycle. ANSWER TO KEY QUESTIons About the Pentose Phosphate Pathway and Gluconeogenesis What is the biochemical basis for favism and how is it related to malarial resistance? Favism is a deficiency in the enzyme glucose-6-phosphate dehydrogenase (G6PD) which is required to maintain flux through the pentose phosphate pathway and provide erythrocytes with NADPH. NADPH is a coenzyme in the glutathione reductase reaction which generates reduced glutathione for cellular detoxification of superoxide radicals by the enzyme glutathione peroxidase. Fava beans are rich in a compound called vicine which is a toxic glycoside that induces oxidative stress in erythrocytes. Individuals with a G6PD deficiency are not able to produce enough NADPH through the pentose phosphate pathway to detoxify vicine, and as a result, suffer from symptoms of diet-induced acute hemolytic anemia, a condition known as favism. It turns out that a productive infection of the mosquito born malarial pathogen Plasmodium requires that erythrocytes have high levels of NADPH to control oxidative stress which would otherwise inhibit the Plasmodium life cycle. Individuals with a G6PD deficiency have naturally reduced levels of NADPH in their
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erythrocytes and are found to be more resistant to malarial infection than normal individuals. This is consistent with this observation that the anti-malarial drug primaquine, which induces oxidative stress in erythrocytes, causes severe hemolytic anemia in individuals with G6PD deficiency. How does fructose-2,6-bisphosphate control flux through gluconeogenesis and glycolysis? Fructose-2,6-bisphosphate (F-2,6-BP) controls flux through gluconeogenesis and glycolysis by reciprocal allosteric regulation of the glycolytic enzyme phosphofructokinase-1 (PFK-1) and the gluconeogenic enzyme fructose-1,6-bisphophatase (FBPase-1). F-2,6-BP is structurally related to fructose-6P and fructose-1,6BP, but is not a metabolic intermediate in either the glycolytic or gluconeogenic pathways. F-2,6-BP is an allosteric activator of PFK-1 activity and an allosteric inhibitor of FBPase-1, therefore in the presence of elevated levels of F-2,6-BP, metabolic flux through glycolysis is much greater than through gluconeogenesis. The amount of F-2,6-BP in the cell is determined by a dual function enzyme that contains a kinase activity called phosphofructokinase-2 (PFK-2) which phosphorylates fructose-6P to form F-2,6-BP, and a phosphatase activity called fructose-2,6-bisphosphatase (FBPase-2) that dephosphorylates F-2,6BP to form fructose-6P. The activity of the PFK-2/FBPase-2 dual function enzyme is controlled by hormone-dependent phosphorylation of a serine residue in the protein. Glucagon signaling through protein kinase A (PKA) results in phosphorylation of the enzyme which inhibits PFK-2 activity and stimulates FBPase-2 activity, thereby leading to decreased levels of F-2,6-BP and increased flux through gluconeogenesis. In contrast, insulin signaling in leads to stimulation of protein phosphatase-1 (PP-1) activity and subsequent dephosphorylation of the PFK-2/FBPase-2 enzyme. This leads to an increase in F-2,6-BP levels which stimulates PFK-1 activity and inhibits FBPase-1 activity, thereby, increasing flux through glycolysis.

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