CHAPTER OUTLINE
Reactions of the Pentose Phosphate Pathway
The PPP and the Control of Oxidative Stress
High-Yield Terms
Glucose-6-phosphate dehydrogenase (G6PDH): primary rate-limiting, oxidative enzyme of the PPP; mutations in this gene are the most common causes of hemolytic anemia worldwide, yet protect individuals from malaria
Transketolase: thiamine-requiring enzyme, enzyme of the nonoxidative stage of the PPP that transfers 2-carbon units
Transaldolase: thiamine-requiring enzyme, enzyme of the nonoxidative stage of the PPP that transfers 3-carbon units
Oxygen burst: phagocytic cells of the immune system utilize NADPH generated via the PPP to generate superoxide radicals to kill phagocytized microorganisms. This requires a dramatic increase in O2 consumption referred to as the oxygen burst
Chronic granulomatous disease: results in individuals harboring defects in the NADPH oxidase system of phagocytic cells, results in frequent pneumonia, abscesses of the skin, tissues, and organs, suppurative arthritis, and osteomyelitis. Defects occur in both the NADPH oxidase complex itself and the enzymes of the PPP that yield the required NADPH, particularly G6PDH
The Pentose Phosphate Pathway
The pentose phosphate pathway (PPP) (also called hexose monophosphate shunt, HMS) consists of series of interconnected reactions that serve as an alternate route for the metabolism of glucose. The pathway is primarily an anabolic pathway that utilizes the 6 carbons of glucose to generate 5-carbon sugars and reducing equivalents in the form of NADPH. However, this pathway does play a role in the oxidation of glucose and under certain conditions can completely oxidize glucose to CO2 and water. The 2 primary functions of this pathway are (1) the generation of NADPH for use in other reductive biosynthetic (anabolic) pathways and (2) to convert glucose carbons into ribose 5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids. Although not a significant function of the PPP, it can operate to metabolize dietary pentose sugars derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates (Figure 15-1).
FIGURE 15-1: Flow chart of pentose phosphate pathway and its connections with the pathway of glycolysis. The full pathway, as indicated, consists of three interconnected cycles in which glucose 6-phosphate is both substrate and end product. The reactions above the broken line are nonreversible, whereas all reactions under that line are freely reversible apart from that catalyzed by fructose 1,6-bisphosphatase. Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
Enzymes that function primarily in the reductive direction utilize the NADP+/NADPH cofactor pair as cofactors as opposed to oxidative enzymes that utilize the NAD+/NADH cofactor pair. The reactions of fatty acid biosynthesis and steroid biosynthesis utilize large amounts of NADPH. As a consequence, cells of the liver, adipose tissue, adrenal cortex, testis, and lactating mammary gland have high levels of the PPP enzymes. In fact 30% of the oxidation of glucose in the liver occurs via the PPP. Additionally, erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the reduction of glutathione (see below). The conversion of ribonucleotides to deoxyribonucleotides, through the action of ribonucleotide reductase, requires NADPH, therefore any rapidly proliferating cell needs large quantities of NADPH.
Although the PPP operates in all cells, with high levels of expression in the above indicated tissues, the highest levels of PPP enzymes (in particular glucose 6-phosphate dehydrogenase) are found in neutrophils and macrophages. These leukocytes are the phagocytic cells of the immune system and they utilize NADPH to generate superoxide radicals from molecular oxygen in a reaction catalyzed by NADPH oxidase. Superoxide anion, in turn, serves to generate other reactive oxygen species (ROS) that kill the phagocytized microorganisms.
Reactions of the Pentose Phosphate Pathway
The reactions of the PPP operate exclusively in the cytoplasm. The PPP is composed of both oxidative and nonoxidative reactions. The oxidation steps, utilizing glucose 6-phosphate (G6P) as the substrate, occur at the beginning of the pathway and are the reactions that generate NADPH. The reactions catalyzed by G6PDH and 6-phosphogluconate dehydrogenase each generate 1 mole of NADPH for every mole of glucose 6-phosphate (G6P) that enters the PPP. The net yield from both of these oxidative reactions is 2 moles of NADPH per mole of G6P (Figure 15-2).
FIGURE 15-2: The pentose phosphate pathway. (P,—PO32−; PRPP, 5-phosphoribosyl 1-pyrophosphate.) Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
The nonoxidative reactions of the PPP are primarily designed to generate R5P. Equally important reactions of the PPP are to convert dietary 5-carbon sugars into both 6- and 3-carbon sugars (fructose 6-phosphate and glyceraldehydes 3-phosphate, respectively) which can then be utilized by the pathways of glycolysis or gluconeogenesis. The primary enzymes involved in the nonoxidative steps of the PPP are transaldolase and transketolase. Transketolase functions to transfer 2-carbon groups from substrates of the PPP, thus rearranging the carbon atoms that enter this pathway. Like other enzymes that transfer 2-carbon groups, transketolase requires thiamine pyrophosphate (TPP) as a cofactor. Transaldolase transfers 3-carbon groups, and thus is also involved in a rearrangement of the carbon skeletons of the substrates of the PPP.
The net result of the PPP is the oxidation of 3 moles of G6P into 3 moles of 5-carbon sugars and 3 moles of CO2. Following rearrangement of the 5-carbon sugars in the nonoxidative reactions 2 moles of G6P are regenerated along with 1 mole of glyceraldehydes 3-phosphate. The G6P can be recycled into the oxidative reactions of the PPP, generating more NADPH. The glyceraldehydes 3-phosphate can be shunted into glycolysis and oxidized to pyruvate or alternatively; it can be utilized by the gluconeogenic enzymes to generate more 6-carbon sugars (fructose 6-phosphate or G6P).
High-Yield Concept
Following exposure to bacteria and other foreign substances there is a dramatic increase in O2 consumption by phagocytes in a phenomenon referred to as the oxygen burst.
The PPP and the Control of Oxidative Stress
Oxidative stress within cells is controlled primarily by the action of the peptide, glutathione (GSH). GSH is a tripeptide composed of γ-glutamate, cysteine, and glycine. The sulfhydryl side chains of the cysteine residues of 2 glutathione molecules form a disulfide bond (GSSG) during the course of being oxidized in reactions with various oxides and lipid hydroperoxides (LOOH) in cells (Figure 15-3).
FIGURE 15-3: Role of the pentose phosphate pathway in the glutathione peroxidase reaction of erythrocytes. (G-SH, reduced glutathione; G-S-S-G, oxidized glutathione; Se, selenium-containing enzyme.) Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil PA. Harper’s Illustrated Biochemistry, 29th ed. New York, NY: McGraw-Hill; 2012.
Oxidative stress also generates peroxides that in turn can be reduced by glutathione to generate water and an alcohol, or two waters if the peroxide is hydrogen peroxide. Regeneration of reduced glutathione (GSH) is carried out by the enzymes glutathione reductase (requiring NADPH) and glutathione peroxidase.
There are at least 3 inborn errors in the PPP that have been identified. The most common being the result of mutations in G6PDH. Extremely rare occurrences of ribose-5-phosphate isomerase and transaldolase deficiency have also been documented. In the transaldolase deficiency individuals with liver problems are the principal symptom in neonates. Any disruption in the level of NADPH production may have a profound effect upon the ability of a cell to deal with oxidative stress (Clinical Box 15-1).
CLINICAL BOX 15-1: CHRONIC GRANULOMATOUS DISEASE
Because of the need for NADPH in phagocytic cells, by the NADPH oxidase system, any defect in enzymes in this process can result in impaired killing of infectious organisms. Chronic granulomatous disease (CGD) is a syndrome that results in individuals harboring defects in the NADPH oxidase system. There are several forms of CGD involving defects in various components of the NADPH oxidase system. Individuals with CGD are at increased risk for specific recurrent infections. The most common are pneumonia; abscesses of the skin, tissues, and organs; suppurative arthritis (invasion of the joints by infectious agent leading to generation of pus); and osteomyelitis (infection of the bone). The majority of patients with CGD harbor mutations in an X-chromosome gene that encodes a component of the NADPH oxidase system. The encoded protein is the β-subunit of cytochrome b245 (gene symbol CYBB), also called p91-PHOX or NOX2. This form of the disorder is referred to as cytochrome b-negative X-linked CGD. There is an autosomal recessive cytochrome b-negative form of CGD due to defects in the α-subunit of cytochrome b245 (gene symbol CYBA), also called p22-PHOX or NOX1. There are also 2 autosomal recessive cytochrome b-positive forms of CGD identified as cytochrome b-positive CGD type I and type II. The type I form is caused by mutation in the neutrophil cytosolic factor 1 (NCF1) gene, which encodes the p47-PHOX (phagocyte oxidase) protein. The type II form is caused by mutation in the NFC2 gene which encodes the p67-PHOX (phagocyte oxidase) protein. Given the role of NADPH in the process of phagocytic killing, it should be clear that individuals with reduced ability to produce NADPH (such as those with G6PDH deficiencies) may also manifest with symptoms of CGD.
The Role of the PPP in the Erythrocyte
The predominant pathways of carbohydrate metabolism in the erythrocyte are glycolysis, the PPP, and 2,3-bisphosphoglycerate (2,3-BPG) metabolism. Glycolysis provides ATP for membrane ion pumps and NADH for reoxidation of methemoglobin (Chapter 6), while the PPP supplies the erythrocyte with NADPH to maintain the reduced state of glutathione. The inability to maintain reduced glutathione in erythrocytes leads to increased accumulation of peroxides, predominantly H2O2, that in turn results in a weakening of the cell wall and concomitant hemolysis. Accumulation of H2O2 also leads to increased rates of oxidation of hemoglobin to methemoglobin that also weakens the cell wall. Glutathione removes peroxides via the action of glutathione peroxidase. The PPP in erythrocytes is essentially the only pathway for these cells to produce NADPH. Any defect in the production of NADPH could, therefore, have profound effects on erythrocyte survival.
Deficiency in the level of activity of G6PDH is the basis of favism, primaquine (an antimalarial drug) sensitivity, and several other drug-sensitive hemolytic anemias, anemia and jaundice in the newborn, and chronic nonspherocytic hemolytic anemia. In addition, G6PDH deficiencies are associated with resistance to the malarial parasite, Plasmodium falciparum, among individuals of Mediterranean and African descent. The basis for this resistance is the weakening of the erythrocyte membrane such that it cannot sustain the parasitic life cycle long enough for productive growth.
REVIEW QUESTIONS
1. Which of the following is a congenital defect in which enzyme may cause hemolytic anemia following administration of the antimalarial drug primaquine?
A. hexokinase
B. pyruvate kinase
C. glucose-6-phosphate dehydrogenase
D. phosphofructokinase
E. glyceraldehyde-3-phosphate dehydrogenase
Answer C: Primaquine is used to treat malaria since it causes lysis of erythrocytes that have been weakened by the presence of the parasite. When administered to humans, primaquine is metabolized into an arylhydroxylamine metabolite that exerts the hemotoxi effects. Primaquine causes methemoglobinemia in all patients. However, dangerous levels of methemoglobinemia only occur in patients with glucose-6-phosphate dehydrogenase (G6PDH) deficiency and, thus, this drug should not be administered to anyone with a deficiency in this enzyme.
2. The transketolase enzyme of the pentose phosphate pathway requires which of the the following for maximal activity?
A. biotin
B. calcium ions
C. coenzyme A
D. tetrahydrofolate
E. TPP
Answer E: Transketolase functions to transfer 2-carbon groups from substrates of the PPP, thus rearranging the carbon atoms that enter this pathway. Like other enzymes that transfer 2-carbon groups, transketolase requires TPP as a cofactor in the transfer reaction.
3. In which of the following tissues would pentose phosphate pathway be expected to be the least active?
A. adipose tissue
B. adrenal gland
C. erythrocytes
D. lactating mammary tissue
E. skeletal muscle
Answer E: The reactions of fatty acid biosynthesis and steroid biosynthesis utilize large amounts of NADPH. As a consequence, cells of the liver, adipose tissue, adrenal cortex, testis, and lactating mammary gland have high levels of the PPP enzymes. Skeletal muscle, although active at de novo fatty acid synthesis, have the lowest levels of expression of enzymes of the PPP.
4. Your patient is suffering from chronic granulomatous disease (CGD) which results in recurrent bouts of infection. CGD is characterized by an inability of phagocytic cells to kill invading microbes that they have engulfed because these cells lack the ability to generate reactive oxygen species such as H2O2. A defect in which of the following enzymes is most likely the cause of the phagocytic cell dysfunction?
A. G6PDH
B. glyceraldehyde-3-phosphate dehydrogenase
C. glycerol-3-phosphate dehydrogenase
D. α-ketoglutarate dehydrogenase
E. pyruvate dehydrogenase
Answer A: The highest levels of PPP enzymes (in particular G6PDH) are found in neutrophils and macrophages. These leukocytes are the phagocytic cells of the immune system and they utilize NADPH to generate superoxide radicals from molecular oxygen in a reaction catalyzed by NADPH oxidase. Therefore, deficiency in the activity of G6PDH can result in defective function in the NADPH oxidase system in these cells.
5. A 22-year-old black male was given the antimalaria drug primaquine to take while on an expedition on the Amazon River. After taking the drug, he developed an acute anemia. The anemia was secondary to an intravascular hemolytic crisis. This crisis was reversed when he discontinued taking the primaquine. This drug-induced hemolytic anemia arises in persons who have a deficiency in which of the following enzymes?
A. glyceraldehyde-3-phosphate dehydrogenase
B. glycerol-3-phosphate dehydrogenase
C. G6PDH
D. pyruvate dehydrogenase
E. succinate dehydrogenase
Answer C: Primaquine is used to treat malaria since it causes lysis of erythrocytes that have been weakened by the presence of the parasite. When administered to humans, primaquine is metabolized into an arylhydroxylamine metabolite that exerts the hemotoxi effects. Primaquine causes methemoglobinemia in all patients. However, dangerous levels of methemoglobinemia only occur in patients with G6PDH deficiency and, thus, this drug should not be administered to anyone with a deficiency of this enzyme.
6. Oxidative breakdown of glucose can occur in certain mammalian cells by mechanisms other than classic glycolysis. In most of these cells, G6P is oxidized to 6-phosphogluconate. Which of the following represents the next intermediate in this alternative glucose oxidation pathway?
A. an aldolase-type split to form glyceric acid and glyceraldehyde 3-phosphate
B. an aldolase-type split to form glycolic acid and erythrose 4-phosphate
C. conversion to 1,6-bisphosphogluconate
D. decarboxylation to produce an aldopentose
E. oxidation to a 6-carbon dicarboxylic acid
Answer D: The next reaction in the PPP, catalyzed by 6-phosphogluconate dehydrogenase, oxidatively decarboxylates 6-phosphogluconate to the 5-carbon aldose sugar, ribulose 5-phopshate.
7. A 2-year-old child who is a native of East Africa has been diagnosed with a deficiency in G6PDH. Following consumption of fava beans the child experiences a mild hemolytic anemia. Which of the following symptoms would also likely be present in this child?
A. coarse facial features and bulging forehead
B. dermatitis, diarrhea, and dementia
C. hypoglycemia and galactosemia
D. ocular stomatitis
E. splenomegaly and jaundice
Answer A: Favism is the term associated with the hemolytic anemia that results from the consumption of fava beans in individuals with deficiencies in G6PDH. The deficiency prevents erythrocytes from coping with the oxidative stress induced by metabolism of substances in fava beans. The continued hemolysis leads to hyperbilirubinemia, splenomegaly, and skeletal abnormalities in infants. The facial anomalies associated with favism are due to repeated episodes of intracranial hemorrhage.
8. Which of the following represents the main function of the pentose phosphate pathway?
A. provides a mechanism for the utilization of the carbon skeletons of excess amino acids
B. provides an alternative pathway for energy production should glycolysis fail
C. provides an alternative pathway for oxidation of excess fructose
D. supplies the cell with hexose sugars and NADH
E. supplies the cell with pentose sugars and NADPH
Answer E: The 2 major functions of the PPP are (1) the generation of reducing equivalents, in the form of NADPH, for reductive biosynthesis reactions within cells and (2) to provide the cell with ribose 5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids.
9. The reactions of the PPP using glucose 6-phosphate as the initial substrate are best described by which of the following statements?
A. they are not required for the production of NADPH in the mature erythrocyte
B. they are required for the metabolism of glucose in the muscles
C. they generate 2 moles of adenosine triphosphate per mole of G6P metabolized to ribulose 5-phosphate.
D. they occur in the matrix of mitochondria
E. they produce 2 moles of NADPH for each mole of CO2 released
Answer E: During the oxidative reactions of the PPP, the enzymes G6PDH and 6-phosphoglucinate dehydrogenase each generate a mole of NADPH. The latter reaction is an oxidative decarboxylation reaction generating the mole of CO2 produced via the PPP.
10. A 50-year-old alcoholic male presents with pain, numbness, tingling, and weakness in his feet. He is diagnosed with thiamine deficiency. Thiamine is required as a cofactor for which of the following enzymes?
A. gluconolactone hydrolase
B. G6PDH
C. 6-phosphogluconate dehydrogenase
D. transaldolase
E. transketolase
Answer D: Transketolase functions to transfer 2-carbon groups from substrates of the PPP, thus rearranging the carbon atoms that enter this pathway. Like other enzymes that transfer 2-carbon groups, transketolase requires TPP as a cofactor in the transfer reaction.
11. Which of the following enzymes represents a key regulated enzyme of the PPP?
A. gluconolactone hydrolase
B. G6PDH
C. 6-phosphogluconate dehydrogenase
D. transaldolase
E. transketolase
Answer B: The key regulated enzyme of the PPP, G6PDH, catalyzes the initial oxidation reaction of the pathway.
12. Xylulose 5-phosphate can be formed from ribulose 5-phosphate via the action of which of the following enzymes?
A. G6PDH
B. phosphopentose epimerase
C. phosphopentose isomerase
D. transaldolase
E. transketolase
Answer B: The conversion of ribulose 5-phosphate to xylulose 5-phosphate is an epimerization reaction. This reaction is catalyzed by the phosphopentose epimerase more commonly called ribulose 5-phosphate epimerase.
13. The PPP is active in liver, adipose tissue, adrenal cortex, thyroid, erythrocytes, testis, and lactating mammary gland. Why is the pathway less active in skeletal muscle?
A. muscle tissue contains very small amounts of the enzymes of the nonoxidative phase of the PPP
B. muscle tissue contains very small amounts of the dehydrogenases of the PPP
C. muscle tissues do not require NADPH
D. the pentose sugars generated by the PPP are not required by muscle tissue
Answer B: The enzymes of the PPP are expressed at highest levels in tissues that carry out high rates of lipid biosynthesis. Skeletal muscle, although active at de novo fatty acid synthesis, does not require high rates of fatty acid synthesis because it can acquire them readily from the blood. Therefore, expression of the oxidative enzymes of the PPP is at low levels in this tissue.
14. While training for a triathlon, a 25-year-old female follows a high-carbohydrate diet that provides for adequate glycogen and fat synthesis. When she begins the competition, fatty acid synthesis stops. The best explanation for this is a decrease in which of the following?
A. gluconeogenesis
B. glycogenolysis
C. oxidative phosphorylation
D. PPP
E. TCA cycle
Answer D: Fatty acid synthesis requires NADPH derived from the PPP via the oxidation of G6P. During strenuous exercise the vast majority of the glucose is shunted into glycolysis for complete oxidation. Therefore, little G6P is available for the PPP.
15. A 32-year-old man of Mediterranean descent is brought to the physician because of a 2-day history of fatigue and shortness of breath. He recently had a urinary tract infection treated with trimethoprim-sulfamethoxazole. His pulse is 100/minute. Physical examination shows pallor and scleral icterus. Laboratory studies show a decreased erythrocyte count and an increased serum bilirubin concentration that is mainly indirect. This patient’s anemia is most likely due to failure of which of the following?
A. bone marrow production of reticulocytes
B. erythrocyte production of lactic acid
C. erythrocyte protection against membrane oxidation
D. reticulocyte maturation
E. reticulocyte synthesis of heme
Answer C: Erythrocytes require a continuous source for NADPH to ensure that oxidized glutathione can be reduced by the glutathione peroxidase system. Glutathione is oxidized when it scavenges reactive oxygen species and by reducing oxidized membrane lipids. Administration of oxidizing drugs such as those of the sulfa class can lead to weakening of erythrocyte plasma membranes due to rapid depletion of reduced glutathione levels and inadequate NADPH-dependent reduction of the oxidized form.
