The pentose phosphate pathway is primarily an anabolic pathway that utilizes the six carbons of glucose to generate five carbon sugars and reducing equivalents. However, this pathway does oxidize glucose and under certain conditions can completely oxidize glucose to CO2 and water. The primary functions of this pathway are:
1. To generate reducing equivalents, in the form of NADPH, for reductive biosynthesis reactions within cells.
2. To provide the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids.
3. 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.
Enzymes that function primarily in the reductive direction utilize the NADP+/NADPH cofactor pair as co-factors 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 as the electron source, 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. Following exposure to bacteria and other foreign substances there is a dramatic increase in O2 consumption by phagocytes. This phenomenon is referred to as the oxygen burst.back to the top
Reactions of the Pentose Phosphate Pathway: The first three reactions of the PPP are referred to as the oxidative portion and are the reaction that yield NADPH. The non-oxidative reactions result in the rearrangement of the carbon skeletons of numerous carbohydrates. G6PDH: glucose-6-phosphate dehydrogenase. PGLS: 6-phosphogluconolactonase. PGD: 6-phosphogluconate dehydrogenase. RPE: ribulose-5-phosphate 3-epimerase. RPIA: ribose-5-phosphate isomerase
The reactions of the PPP operate exclusively in the cytoplasm. From this perspective it is understandable that fatty acid synthesis (as opposed to oxidation) takes place in the cytoplasm. The pentose phosphate pathway has both an oxidative and a non-oxidative arm. 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 glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (PGD) both generate one mole of NADPH for every mole of glucose-6-phosphate that enters the PPP. The conversion of 6-phosphogluconolactone to 6-phosphogluconate is catalyzed by 6-phosphogluconolactonase (PGLS).
The glucose-6-phosphate dehydrogenase gene (symbol: G6PD) is located on the X chromosome (Xq28) and is composed of 14 exons that generate two alternatively spliced mRNAs. These two mRNAs encode the G6PDH isoform a (545 amino acids) and isoform b (515 amino acids) enzymes. The G6PDH isoform a protein is inactive in the native full-length form but may undergo processing to the smaller 515 amino acid active form of the enzyme. The second enzyme of the PPP, 6-phosphogluconate dehydrogenase is encoded by the phosphogluconate dehydrogenase gene (symbol: PGD). The PGD gene is located on chromosome 1p36.33 and is composed of 13 exons that generate several alternatively spliced mRNAs that encode three different sized isoforms of 6-phosphogluconate dehydrogenase.
The non-oxidative reactions of the PPP are primarily designed to generate ribose-5-phosphate (R5P). Other reactions of the PPP that are of less physiological significance are designed to convert dietary five carbon sugars into both six (fructose-6-phosphate) and three (glyceraldehyde-3-phosphate) carbon sugars which can then be utilized by the pathways of glycolysis. The primary enzymes involved in the non-oxidative steps of the PPP are transaldolase and transketolase. Transketolase functions to transfer two-carbon groups from substrates of the PPP thus, rearranging the carbon atoms that enter this pathway. Like other enzymes that transfer two-carbon groups, transketolase requires thiamine pyrophosphate (TPP) as a co-factor in the transfer reaction. Two facts regarding transketolase make it a diagnostically useful enzyme. The enzyme is expressed at high levels in red blood cells, which are easy to isolate and analyze, and the only vitamin-derived cofactor it requires is TPP. Therefore, assay for reduced activity of this enzyme, in red blood cell lysates, is highly diagnostic in cases of suspected thiamine deficiency. Transaldolase transfers three-carbon groups and thus is also involved in a rearrangement of the carbon skeletons of the substrates of the PPP. The transaldolase reaction involves Schiff base formation between the substrate and a lysine residue in the enzyme.
Transketolase is encoded by the TKT gene which is located on chromosome 3p14.3 and is composed of 16 exons that generate several alternatively spliced mRNAs. Transaldolase is encoded by the TALDO1 gene which is located on chromosome 11p15.5-p15.4 and is composed of 8 exons that encode a protein of 337 amino acids. A molecularly interesting fact about the TALDO1 gene is that exons 2 and 3 were derived as a result of the insertion of a retrotransposon into the gene.
There are two transketolase-related genes in the human genome. One is found on the X chromosome (Xq28) and is identified as the transketolase-like 1 gene (symbol: TKTL1). The other gene (symbol: TKTL2) is located on chromosome 4q32.2. The TKTL1 encoded protein lacks 38 amino acids, compared to the TKT protein, in the TPP-binding region. All TPP-dependent enzymes contain a highly similar TPP-binding domain and its lack in the TKTL1 protein strongly suggests that it is unlikely that TKTL1 is a TPP-dependent protein capable of catalyzing the transketolase reaction. Indeed, recent evidence has confirmed that the TKTL1 protein does not catalyze the transketolase reaction of the PPP. Intense interest in the TKTL1 gene, and its encoded protein, was stimulated because it was shown that the level of TKTL1 expression correlated with poor patient outcomes and metastasis in many solid tumours. In addition, specific inhibition of TKTL1 mRNA has been shown to inhibit cancer cell proliferation in functional studies.
The net result of the PPP, if not used solely for R5P production, is the oxidation of G6P, a six-carbon sugar, into a five-carbon sugar. In turn, three moles of five-carbon sugar are converted, via the enzymes of the PPP, back into two moles of six-carbon sugars and one mole of three-carbon sugar. The six-carbon sugars can be recycled into the pathway in the form of G6P, generating more NADPH. The three-carbon sugar generated is glyceraldehyde-3-phosphate which can be shunted to glycolysis and oxidized to pyruvate. Alternatively, it can be utilized by the gluconeogenic enzymes to generate more six-carbon sugars, fructose-6-phosphate or glucose-6-phosphate.back to the top
Oxidative stress within cells is controlled primarily by the action of the peptide, glutathione, GSH. See Specialized Products of Amino Acids for the synthesis of GSH. GSH is a tripeptide composed of γ-glutamate, cysteine and glycine. The sulfhydryl side chains of the cysteine residues of two glutathione molecules form a disulfide bond (GSSG) during the course of being oxidized in reactions with various oxides and peroxides in cells. Reduction of GSSG to two moles of GSH is the function of glutathione reductase, an enzyme that requires coupled oxidation of NADPH.
The cysteine thiol of GSH plays the role in reducing oxidized thiols in other proteins. Oxidation of two cysteine thiols forms a disulfide bond. Although this bond plays a very important role in protein structure and function, inappropriately introduced disulfides can be detrimental. Glutathione can reduce disulfides nonenzymatically. 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 were hydrogen peroxide. Regeneration of reduced glutathione is carried out by the enzyme, glutathione reductase. This enzyme requires the co-factor NADPH when operating in the direction of glutathione reduction which is the thermodynamically favored direction of the reaction.
There are at least three inborn errors in the pentose phosphate pathway that have been identified. The most common being the result of mutations in glucose-6-phosphate dehydrogenase (G6PDH). Extremely rare occurrences of ribose-5-phosphate isomerase and transaldolase deficiency have also been documented. In the transaldolase deficiency individuals with liver problems were the principal symptom in neonates. It should be clear that any disruption in the level of NADPH may have a profound effect upon a cells ability to deal with oxidative stress. No other cell than the erythrocyte is exposed to greater oxidizing conditions. After all it is the oxygen carrier of the body.back to the top
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 two 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.back to the top
The predominant pathways of carbohydrate metabolism in the red blood cell (RBC) are glycolysis, the PPP and 2,3-bisphosphoglycerate (2,3-BPG) metabolism (refer to discussion of hemoglobin for review of the synthesis and role role of 2,3-BPG). Glycolysis provides ATP for membrane ion pumps and NADH for re-oxidation of methemoglobin. The PPP supplies the RBC with NADPH to maintain the reduced state of glutathione. The inability to maintain reduced glutathione in RBCs leads to increased accumulation of peroxides, predominantly H2O2, that in turn results in a weakening of the cell wall as a result of membrane lipid peroxidation resulting in concomitant hemolysis. Accumulation of H2O2 also leads to increased rates of cysteine sulfhydryl oxidation in hemoglobin resulting in the formation of cross-linked complexes of denatured hemoglobin. These large complexes of denatured hemoglobin can be visualized by light microscopy and are referred to as Heinz bodies. Glutathione removes peroxides from membrane lipids and serves as a substrate for H2O2 reduction to H2O via the action of glutathione peroxidase. The PPP in erythrocytes is the only pathway for these cells to produce NADPH, therefore, any defect in the production of NADPH, such as due to deficiencies in glucose-6-phosphate dehydrogenase, will have profound effects on erythrocyte survival.
Pathway for reactive oxygen species (ROS) removal in erythrocytes. Formation of the reactive oxygen species (ROS), H2O2, can occur spontaneously from O2 or is generated via superoxide dismutase (SOD) action on superoxide anion produced as a result of O2 oxidation of the ferrous iron (Fe2+) to ferric iron (Fe3+) in hemoglobin. The consequence of the latter reaction is the formation of methemoglobin Since hemoglobin is a heterotetramer, and each subunit contains Fe2+ iron in its heme there is the potential for multiple Fe3+ irons to be present in methemoglobin. The Fe3+ form of iron does not bind O2, however, the presence of at least one Fe3+ in the hemoglobin tetramer results in enhanced binding of the O2 to the remaining Fe2+ irons causing reduced delivery of the O2 to the tissues with potential for cyanosis. Normal daily levels of methemoglobin range from 0.5%–3%. The ferric iron in methemoglobin is reduced to ferrous via the action of the NADH-requiring enzyme, methemoglobin reductase (cytochrome b5 reductase 3: CYB5R3). The NADPH produced in the PPP, and the antioxidant glutathione (GSH), are both necessary for the continual removal of ROS from within the erythrocyte (red blood cell, RBC). The enzyme, glutathione peroxidase (GPx) utilizes reduced glutathione (GSH) as the electron donor in the process of reducing H2O2 to H2O while simultaneously generating oxidized glutathione (GS-SG). GPx is one of several selenocysteine-containing red-ox enzymes. In order for continued use of GSH, the oxidized molecule needs to be reduced via the action of the NADPH-requiring enzyme, glutathione reductase.
The glutathione reductase gene (symbol: GSR) is located on chromosome 8p21.1 and is composed of 13 exons that generate four alternatively spliced mRNAs that encode four distinct protein isoforms. Humans express eight distinct glutathione peroxidase genes identified as GPX1–GPX8. The GPX1 gene is located on chromosome 3p21.3 and is composed of 2 exons that generate two alternatively spliced mRNAs encoding two distinct protein isoforms. The GPX2 gene is located on chromosome 14q24.1 and is composed of 4 exons that encode a 190 amino acid protein. The GPX3 gene is located on chromosome 5q33.1 and is composed of 5 exons that encode a 226 amino acid precursor protein. The GPX4 gene is located on chromosome 19p13.3 and is composed of 8 exons that generate three alternatively spliced mRNAs that encode several distinct protein isoforms that exhibit distinct subcellular sites of localization including the nucleus, the mitochondria, and the cytosol. In addition, one of the GPX4 encoded proteins plays a role in sperm maturation, a process that is structural not enzymatic. The GPX5 gene is located on chromosome 6p22.1 and is composed of 7 exons that generate two alternatively spliced mRNAs encoding two protein isoforms. The GPX5 encoded mRNAs do not contain a seleocysteine (UGA) codon and, therefor, encode selenium-independent enzymes. Expression of the GPX5 gene is restricted to the epididymis in the male gonads and the encoded proteins function to protect the membranes of spermatozoa during their maturation. The GPX6 gene is located on chromosome 6p22.1 near the GPX5 gene and is composed of 5 exons that encode a 221 amino acid precursor protein. Expression of the GPX6 gene is restricted to the olfactory epithelium. The GPX7 gene is located on chromosome 1p32 and is composed of 3 exons that encode a 187 amino acid precursor protein. The GPX8 gene is located on chromosome 5q11.2 and is composed of 3 exons that encode a putative GPx enzyme.
Deficiency in the level of activity of glucose-6-phosphate dehydrogenase (G6PDH) is the basis of favism, primaquine (an anti-malarial drug) sensitivity and some 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 red cell membrane (the erythrocyte is the host cell for the parasite) such that it cannot sustain the parasitic life cycle long enough for productive growth.back to the top