13.4: Pentose Phosphate Pathway of Glucose Oxidation

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Learning Goals

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Products, Purpose, and Overall Logic of the Pentose Phosphate Pathway

• Explain why the pentose phosphate pathway is an essential complement to glycolysis, identifying its two major products (NADPH and ribose-5-phosphate), the metabolic demands each satisfies, and why the pathway produces no ATP.
• Describe how NADPH generated by the oxidative branch maintains cellular redox balance, with particular emphasis on its role in regenerating reduced glutathione and protecting erythrocytes — which lack mitochondria — from oxidative damage by ROS.

Oxidative Branch: Enzymes and Mechanisms

• Trace the three enzymatic steps of the oxidative branch from glucose-6-phosphate to ribulose-5-phosphate, explaining the chemical transformations (oxidation, hydrolysis, and oxidative decarboxylation) and the production of two NADPH molecules per glucose-6-phosphate consumed.
• Explain why glucose-6-phosphate dehydrogenase (G6PD) is the committed and rate-limiting enzyme of the oxidative branch, how it is regulated by the NADP⁺/NADPH ratio, and why G6PD deficiency causes hemolytic anemia under oxidative stress conditions such as infection or ingestion of fava beans.

Non-Oxidative Branch: Enzymes and Mechanisms

• Describe the roles of ribose-5-phosphate isomerase and ribulose-5-phosphate epimerase in interconverting ribulose-5-phosphate with ribose-5-phosphate and xylulose-5-phosphate, and explain how these reactions supply the substrates for transketolase and transaldolase.
• Explain the carbon-transfer logic of transketolase (5C + 5C → 3C + 7C; 5C + 4C → 3C + 6C) and transaldolase (3C + 7C → 4C + 6C), including the mechanistic role of TPP in transketolase and the Schiff base intermediate in transaldolase, and identify the glycolytic intermediates that the non-oxidative branch generates.

Regulation and Metabolic Integration

• Explain how the cell independently adjusts the relative outputs of NADPH and ribose-5-phosphate by modulating flux through the oxidative and non-oxidative branches in response to changing biosynthetic and redox demands, and describe how tissue-specific differences in PPP activity reflect the metabolic priorities of different cell types.

Introduction

The pentose phosphate pathway (PPP), or the pentose phosphate shunt, is an important part of glucose metabolism. The PPP branches after the first step of glycolysis and consumes the intermediate glucose 6-phosphate (G6P) to generate fructose 6-phosphate (F6P) and glyceraldehyde 3-phosphate (G3P) through the oxidative and non-oxidative branches of the PPP. Unlike glycolysis and aerobic glucose oxidation, the PPP does not provide adenosine 5′-triphosphate (ATP) to meet cells' energy demands. Instead, it supplies NADPH and ribose 5-phosphate (R5P). These two metabolites are vital for cell survival and proliferation. R5P is a building block for nucleic acid synthesis. NADPH offers the reducing power required to synthesize fatty acids, sterols, nucleotides, and non-essential amino acids. Moreover, the NADPH-derived conversion of oxidized glutathione (GSSG) to reduced glutathione (GSH) via glutathione reductase is important for cellular antioxidant defenses. Interestingly, NADPH also serves as the substrate of NADPH oxidases (NOXs), which produce reactive oxygen species (ROS).

Both the oxidative branch and non-oxidative branch of the PPP take place in the cytosol (Figure \(\PageIndex{1}\)). Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the oxidative PPP, determining the flux of G6P into the pathway. G6PD catalyzes the conversion of G6P to 6-phosphogluconolactone, producing NADPH. 6-phosphogluconolactonase (6PGL) is the enzyme that hydrolyzes 6-phosphogluconolactone to produce 6-phosphogluconate (6PG). 6-phosphogluconate dehydrogenase (6PGD) converts 6-PG to ribulose 5-phosphate (Ru5P) and generates NAPDH (Figure \(\PageIndex{1}\)). The largest source of cytosolic NADPH in mammalian cells is the oxidative PPP.

The non-oxidative branch consists of a series of reversible transfer reactions of chemical groups. Ribose-5-phosphate isomerase (RPI) and ribulose-5-phosphate epimerase (RPE) catalyze reversible reactions that convert Ru5P to R5P and xylulose-5-phosphate (Xu5P), respectively. TKT catalyzes two reversible reactions. One is the conversion of Xu5P and R5P to G3P and sedoheptulose 7-phosphate (S7P). The other is the conversion of Xu5P and erythrose 4-phosphate (E4P) to G3P and F6P. Therefore, TKT can bi-directionally regulate the carbon flux between the non-oxidative PPP and glycolysis or gluconeogenesis. Transaldolase (TALDO) reversibly converts G3P and S7P to E4P and F6P. The non-oxidative branch not only replenishes metabolites of the oxidative branch (by their reversal), but also regulates the flux of glycolysis or gluconeogenesis by providing F6P and G3P (Figure \(\PageIndex{1}\)).

Figure \(\PageIndex{1}\): The pentose phosphate pathway (PPP). The PPP branches after the first step of glycolysis, returning to fructose-6-phosphate and glyceraldehyde-3-phosphate in the glycolytic and gluconeogenic pathways. The PPP produces R5P and NADPH for biosynthesis and redox regulation. Enzymes in the oxidative and non-oxidative PPP are shaded in green.  Figure from: Ge, T. et al. (2020) Cellular Endocrinology DOI:10.3389

Oxidative branch

The oxidative branch of PPP (ox-PPP) is a non-reversible metabolic pathway where glucose-6-phosphate (G6P) is transformed into 6-phosphoglucono-δ-lactone by glucose-6-phosphate dehydrogenase (G6PD) and, subsequently, to ribulose-5-phosphate by 6-phosphogluconate dehydrogenase (6PGD) with the concomitant production of nicotinamide adenine dinucleotide phosphate (NADPH). The resulting ribulose-5-phosphate is then converted to ribose-5-phosphate and used for the biosynthesis of nucleotides (Figure\(\PageIndex{2}\)).

Figure \(\PageIndex{2}\): Summary of the oxidative branch of phosphopentose pathway

The first enzyme in the oxidative branch is glucose-6-phosphate dehydrogenase. It is also the first committed enzyme in the pathway and plays a regulatory role. PPP metabolizes 5-30% of glucose, depending on tissue type. The enzymatic reaction is:

1. Glucose-6-phosphate dehydrogenase: Glc6P + NADP⁺ → 6-phosphogluconolactone + NADPH

The oxidative branch produces NADPH for reductive biosynthesis, and it also maintains the reducing condition of the cell to protect it against oxidative stress, which is especially important in erythrocytes (Figure\(\PageIndex{3}\)). It also initiates the pathway for the production of 5-carbon sugars for nucleotide biosynthesis.

G6PDH and ROS

G6PDH is very important for protecting red blood cells against free radicals, since they lack mitochondria that could provide another source of NADPH. NADPH is involved in protection against ROS through the three cycles shown in Figure \(\PageIndex{3}\).

Figure \(\PageIndex{3}\): Function of G6PD enzyme in the PPP from red blood cells. In G6PD-normal red cells, the NADPH is produced by the action of glucose 6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) enzymes. The NADPH serves as a proton donor to regenerate the GSSG. Cat = Catalase; GPx = Glutathione peroxidase; GR = Glutathione reductase; G6PD = glucose 6-phosphate dehydrogenase; 6PGL = 6-phosphogluconolactonase; 6GPD = 6-phosphogluconate dehydrogenase; SOD = Superoxide dismutase; GSH = Reduced glutathione; GSSG = Oxidized glutathione; H₂O₂ = Peroxide; O₂⁻ = Superoxide. Gomez-Manzo et al. Int J Mol Sci. 2016 Dec; 17(12): 2069. doi: 10.3390/ijms17122069. Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

NADPH can maintain glutathione in its reduced form, which, as a substrate for catalase, can help rid the cell of hydrogen peroxide and, indirectly, other ROS.

The enzyme is active as a dimer or tetramer. Each monomer in the complex has a substrate binding site that binds G6P and a catalytic coenzyme-binding site that binds NADP⁺/NADPH. For some higher organisms, such as humans, G6PD contains an additional NADP⁺ binding site, called the NADP⁺ structural site, that does not seem to participate directly in the reaction catalyzed by G6PD. The evolutionary purpose of the NADP⁺ binding site is unknown; however, it does contribute to the overall stability of the enzyme.

Mutations that cause dysfunction or deficiencies are very common, especially in males and in Africa, Asia, the Mediterranean, and the Middle East, in a geographic distribution that parallels the incidence of malaria. Interestingly, many mutations occur near the NADP⁺ binding site. Glucose-6-phosphate dehydrogenase deficiency is very common worldwide and can cause acute hemolytic anemia in response to simple infections, ingestion of fava beans, or reactions to certain medicines.

An abbreviated mechanism for glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides is shown in Figure \(\PageIndex{4}\).

Figure \(\PageIndex{4}\): Mechanism of Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase. Ribeiro AJM et al. (2017), Nucleic Acids Res, 46, D618-D623. Mechanism and Catalytic Site Atlas (M-CSA): a database of enzyme reaction mechanisms and active sites. DOI:10.1093/nar/gkx1012. PMID:29106569 https://www.ebi.ac.uk/thornton-srv/m-csa/entry/843/. Creative Commons Attribution 4.0 International (CC BY 4.0) License.

Figure \(\PageIndex{5}\) shows an interactive iCn3D model of the glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides (1DPG).

Figure \(\PageIndex{5}\): Glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides (1DPG). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...m8NCXiBuQbTQb7

Interestingly, this bacterium has an incomplete glycolytic pathway and can use this enzyme with either NADP⁺ for anabolism or NAD⁺ for catabolism.

Each monomer can bind two NADP⁺, one at a site that promotes structural integrity and stability, and the other at the catalytic site where NADP⁺ serves as a substrate (or cofactor). The other substrate, glucose-6-phosphate, binds between the two.

Figure \(\PageIndex{6}\) shows an interactive iCn3D model of the human glucose 6-phosphate dehydrogenase with bound structural and substrate NADP⁺ (2BH9).

Figure \(\PageIndex{6}\): Human glucose 6-phosphate dehydrogenase with bound structural and substrate NADP (2BH9).(Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?NFJnS7UtUt3Uwu2D7

The biological unit shown is a dimer. The structural NADP⁺ is shown in spacefill bound in the N-terminal end of each monomer, while the substrate (cofactor) NADP⁺ is shown in sticks. Arg 459 is shown in spacefill.

A common disease-causing mutation of G6PDH is the Canton R459L mutation. It's common in China and Southeast Asia. The enzyme's activity is significantly decreased, and the enzyme is less able to form tetramers. It unfolds at a lower temperature. This suggests that the mutation causes a significant conformation change. Arg459 is shown in the model above. Its contribution to interhelical noncovalent interactions with D181 and N185. D181 is shown in proximity to Arg459 in the above model.

Figure \(\PageIndex{7}\)s shows a static image of glucose 6-phosphate dehydrogenase with bound structural and substrate NADP⁺ as well as glucose-6-phosphate.

Figure \(\PageIndex{7}\): Glucose 6-phosphate dehydrogenase with bound structural and substrate NADP⁺ as well as glucose-6-phosphate (2BHL and 2BH9). Structural NADP⁺ (blue molecular surface), catalytic NADP⁺ (dark purple molecular surface), and G6P substrate (yellow molecular surface) in the dimer are shown. The two monomers are shown in cyan and green. Right inset, close-up of the dimer interface and both structural NADP⁺ molecules. Gomez-Manzo et al., ibid.

The second reaction of the oxidative branch is mediated by the phosphogluconolactonase (6PGL, PGLS) enzyme. The overall reaction is shown here:

2. 6-phosphogluconolactamase: 6-phosphogluconolactone + H₂O → 6-phosphogluconate

6PGL is a cytosolic enzyme found in all organisms that catalyzes the hydrolysis of 6-phosphogluconolactone to 6-phosphogluconic acid in the oxidative phase of the pentose phosphate pathway. 6PGL hydrolysis of 6-phosphogluconolactone to 6-phosphogluconic acid has been proposed to proceed via proton transfer to the O5 ring oxygen atom (Figure \(\PageIndex{8}\)). The reaction is initiated via the attack of a hydroxide ion at the C5 ester. A tetrahedral intermediate forms, and the elimination of the ester linkage follows, aided by the donation of a proton from an active site histidine residue.

Figure \(\PageIndex{8}\): Mechanism for 6-phosphogluconolactamase.  His 165 and Asp 163 appear involved in a proton relay scheme. His 163 is conserved in lactonase. Arg 77 and 200 are involved in binding the substrate.

Figure \(\PageIndex{9}\) shows an interactive iCn3D model of the 6-phosphogluconolactonase from Trypanosoma brucei complexed with 6-phosphogluconic acid (3E7F). This enzyme is a target for developing drug treatment strategies for African sleeping sickness.

Figure \(\PageIndex{9}\): 6-phosphogluconolactonase from Trypanosoma brucei complexed with 6-phosphogluconic acid (3E7F). The key residues involved in binding and the charge-relay system are shown in sticks and labeled. The product, 6-phosphogluconic acid, is shown in spacefill. The single sphere is a Zn^2+, which does not appear to be involved in catalysis. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?jRgi1nfrSGGgTYWo8

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Glyoxosomes and glycosomes

Trypanosomiasis is a disease commonly known as African human trypanosomiasis, or African sleeping sickness. This infectious disease is caused by the parasites Trypanosoma brucei gambiense or Trypanosoma brucei rhodesiense, which are transmitted by the tsetse fly.

The tsetse fly vector, Glossina, carries the trypanosome in the midgut after a blood meal. These protozoa then migrate to the salivary glands of the fly, which can be transmitted during the next feeding. After inoculation into the host, the parasite can live freely in the bloodstream and evade mammalian host defenses through variable surface glycoproteins (VSGs). The clinical disease has two stages. These are characterized by an early/first hemolymphatic stage and a late/second meningoencephalitis stage, with invasion of the central nervous system (CNS). In stage 1, systemic symptoms develop, including intermittent fever, headache, pruritus, and lymphadenopathy. Undulating fevers reflect parasites multiplying within the blood. Less frequent hepatosplenomegaly may occur in the early stage. In the late/second stage, CNS symptoms manifest as sleep disturbances or neuropsychiatric disorders. A sleep disorder is the most common symptom of the second stage, and it is from this that the term “African sleeping sickness” was ascribed.

New Drug Targets:

Enzymes involved in the pentose phosphate pathway offer a new set of drug targets to combat parasitic infections, such as those caused by trypanosomes. In this context, new therapeutics have focused on disrupting glycosomes in these organisms. Glyoxosomes are specialized peroxisomes found in plants and some fungi. They contain enzymes for the glyoxylate shunt and can therefore produce some glucogenic intermediates. In trypanosomes and Leishmania, another internal organelle, the glycosome, is found. This houses most of the glycolytic enzymes and also the oxidative enzymes of the pentose phosphate pathway. This makes the enzymes in the glycosome unique drug targets.

The third enzyme utilized in the oxidative branch of the PPP is 6-phosphogluconate dehydrogenase (6PGDH). The overall reaction is shown here:

3. 6-phosphogluconate dehydrogenase: 6-phosphogluconate + NADP⁺ ↔ ribulose-5-phosphate + NADPH + CO₂

6PGDH catalyzes a reversible oxidative decarboxylation reaction, as shown in Figure \(\PageIndex{10}\). Oxidation, followed by decarboxylation, forms an enediol intermediate, which is then converted to the product. Notably, NADP⁺ serves as an electron acceptor in the reaction, producing a second molecule of NADPH. Essentially, an active-site lysine abstracts a proton from the substrate, 6-phosphogluconate, and NADP⁺ is reduced, forming a ketone intermediate. Decarboxylation leads to the formation of the enediol intermediate. The active site lysine abstracts a proton during the second half of the reaction, leading to the formation of ribulose 5-phosphate. The abstracted proton is transferred to the active site Glu190, resetting the enzyme for another catalytic cycle.

 

Figure \(\PageIndex{10}\): Mechanism of 6-phosphogluconate dehydrogenase.

Figure \(\PageIndex{11}\) shows an interactive iCn3D model of sheep 6-phosphogluconate dehydrogenase (2PGD).

Figure \(\PageIndex{11}\): Sheep 6-phosphogluconate dehydrogenase (2PGD). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...DqosWmFg1DqK16

The protein is a homodimer in which the monomers act independently: each contains a large, mainly alpha-helical domain and a smaller beta-alpha-beta domain, containing a mixed parallel and anti-parallel 6-stranded beta-sheet. NADP⁺ is bound in a cleft in the small domain, and the substrate binds in an adjacent pocket.

Nonoxidative branch

The non-oxidative branch of the pentose phosphate pathway (nonox-PPP) is responsible for forming simple sugars within the cell and maintaining these building blocks in appropriate concentrations. The enzymes utilized in this pathway mediate a set of reversible reactions that lead to the production of ribose-5-phosphate, xylulose-5-phosphate, sedoheptulose 7-phosphate, and erythrose 4-phosphate, as well as intermediates utilized in the glycolytic pathway, including glyceraldehyde-3-phosphate and fructose-6-phosphate. Major enzymes utilized include transketolase (TKT) and transaldolase (TALDO), as well as important isomerase and epimerase enzymes.

Ribose 5-phosphate is an important building block for the biosynthesis of nucleotides, and erythrose 4-phosphate is used to synthesize aromatic amino acids. When these simple sugars are in excess, they can also be converted into glycolytic intermediates and utilized for energy production.

The nonoxidative branch of the phosphopentose pathway is shown in Figure \(\PageIndex{12}\).

Figure \(\PageIndex{12}\): Nonoxidative branch of the phosphopentose pathway

Now, let's examine the individual enzymes. Ribulose 5-phosphate is the starting place for the non-oxidative portion of the PPP. It can be converted down two different pathways, either to ribose-5-phosphate or to xylulose-5-phosphate. Ribose-5-phosphate isomerase is involved in the ketose-aldohexose conversion and will be the first enzyme discussed.

Ribose-5-phosphate isomerase (Rpi)

This enzyme is fully reversible and is usually referred to as ribose-5-phosphate isomerase. However, it is also known as ribulose-5-phosphate isomerase, as it mediates the interconversion between this aldohexose-ketohexose pair. In addition to the PPP, this enzymatic reaction is also required in the Calvin cycle in photosynthesis.

There are two different Rpi enzymes, RpiA and RpiB, with little sequence or structural similarity and distinct mechanisms. RpiA is found in all three kingdoms of life and is highly conserved due to its role in the PPP and the Calvin Cycle of photosynthesis. RpiB, on the other hand, is only found in some bacteria and protozoans. Thus, RpiB is a potential therapeutic target for treating diseases such as African Sleeping Sickness, Chagas disease, and leishmaniasis. Although RpiA and RpiB are structurally very different, both enzymes catalyze the isomerization reaction via an enediol intermediate, using the linear form of the sugar. The reaction mechanism of RpiB is detailed below.

Figure \(\PageIndex{13}\) shows a mechanism for Escherichia coli RpiB.

Figure \(\PageIndex{13}\): Mechanism for Escherichia coli RpiB. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/680/. Creative Commons Attribution 4.0 International (CC BY 4.0) License.

RpiB is a homodimer that can form a tetramer. The monomer subunits are coordinated in a head-to-tail format, such that there are two active sites per dimer where side chains from each monomer contribute to each active site (Figure \(\PageIndex{14}\)). The reaction mechanism requires two major steps: (1) ring opening of the furanose form of the sugar, and (2) isomerization through an enediol intermediate (Figure \(\PageIndex{13}\)). Figure \(\PageIndex{14}\) shows an interactive iCn3D model of Ribose-5-phosphate isomerase (RPIB_AlsB) from Escherichia coli (1NN4).

Figure \(\PageIndex{14}\): Ribose-5-phosphate isomerase (RPIB_AlsB) from Escherichia coli (1NN4).Active site residues are indicated in each of the monomer subunits. (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ByuLUqhhs9B8c9

The other major pathway for ribulose 5-phosphate conversion involves the ribulose 5-phosphate (3) epimerase (RPE) and the conversion to xylulose 5-phosphate.

Ribulose-5-phosphate (3) epimerase (RPE)

This enzyme is also called pentose-5-phosphate 3-epimerase (PPE). Sugar epimerases alter the stereochemistry of the sugar at one of its alcohol positions, creating a different epimer. The RPE enzyme has a Zn²⁺ cofactor; however, this cofactor is not essential for enzyme activity. A Zn²⁺ independent form can still function and stabilize an oxyanion intermediate with adjacent and conserved methionines. A mechanism of the Zn²⁺-dependent form of the enzyme is shown in Figure \(\PageIndex{15}\).

Figure \(\PageIndex{15}\): Mechanism for rice ribulose-5-phosphate epimerase

The enzyme utilizes an acid-base catalytic mechanism to mediate the formation of a trans-2,3-enediol phosphate intermediate. Key aspartic acid residues act as proton donors and acceptors during the reaction. A zinc metal cofactor helps stabilize charges during the reaction.

Figure \(\PageIndex{16}\) shows an interactive iCn3D model of cytosolic D-ribulose-5-phosphate 3-epimerase from rice (1h1z).

Figure \(\PageIndex{16}\): Cytosolic D-ribulose-5-phosphate 3-epimerase from rice (1h1z). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...AH7uS2T8hsnpXA

Transketolase (TK)

The pool of ribose 5-phosphate created can be utilized for nucleotide production, or some of it can react with the xylulose 5-phosphate created in the RPE reaction. The transketolase enzyme converts two five-carbon sugars (ribose 5-phosphate and xylulose 5-phosphate) into a 3-carbon and a 7-carbon sugar (glyceraldehyde 3-phosphate and sedoheptulose 7-phosphate). Transketolase enzymes transfer ketone functional groups from ketoses to aldoses, effectively creating a new ketose that is two-carbon atoms larger. The ketose donor then becomes an aldose with two fewer carbons. The basic transketolase reaction shown in Figure \(\PageIndex{17}\)

Figure \(\PageIndex{17}\): Transketolase reaction. The reaction shows the reversible conversion of ribose 5-phosphate and xylulose 5-phosphate to sedoheptulose 7-phosphate and glyceraldehyde 3-phosphate.

Note the number of carbons in the reactants and products: 5C + 5C ↔ 3C + 7C. For this reversible reaction, the enzyme uses the cofactor thiamine pyrophosphate (TPP) and a divalent cation. The enzyme transfers a 2C ketol group from xylulose-5-phosphate to ribose-5-phosphate, an aldose. Glyceraldehyde-3-phosphate, the other product, is a glycolytic intermediate that can be used in the glycolytic pathway.

The mechanism for the reverse reaction (yeast numbering system) is shown in Figure \(\PageIndex{18}\). In this reaction, sedoheptulose 7-phosphate binds with the enzyme, and the TPP cofactor is activated to form a carbanion. The carbanion mediates nucleophilic attack on the carbonyl carbon of the substrate, forming a covalent intermediate. His263 serves as a base and abstracts a proton, enabling bond cleavage and the formation of ribose 5-phosphate. Ribose 5-phosphate leaves the active site, and glyceraldehyde 3-phosphate enters. The two-carbon intermediate (covalently bound to the TPP) mediates nucleophilic attack on the glyceraldehyde 3-phosphate, enabling the formation of xylulose 5-phosphate and the restoration of the enzyme.

Figure \(\PageIndex{18}\): Mechanism of the transketolase reaction during the pentose phosphate pathway.
https://www.ebi.ac.uk/thornton-srv/m-csa/entry/219/

Figure \(\PageIndex{19}\) shows an interactive iCn3D model of Human transketolase in a covalent complex with donor ketose D-xylulose-5-phosphate (4kxv).

Figure \(\PageIndex{19}\): Human transketolase in covalent complex with donor ketose D-xylulose-5-phosphate (4kxv). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...WtGtQ7mKA6UYx7

The enzyme is a dimer (gray and cyan coloring). Numbering of key residues compared to the yeast mechanism:

Human	Yeast
E366	E418
Q428	H481
H258	H262
H38	H30

Also shown are the human active-site residues H474, R318, and S345. The covalent xylulose-5-phosphate adduct is shown in spacefill and labeled DX5 (xylitol-5-phosphate). TPP is shown in sticks and labeled. The metal ion is Ca²⁺. Structural analyses show a 20⁰ distortion in the planarity of the cofactor-substrate bond and a lengthening of the C-C bond in the substrate, which breaks.

Transaldolase

In addition to being used as a resource in the glycolytic pathway, glyceraldehyde 3-phosphate can also serve as a substrate in a transaldolase reaction, along with the sedoheptulose 7-phosphate produced in the previous reaction. This results in the formation of erythrose-4-phosphate and fructose-6-phosphate. A summary of the reaction is shown in Figure \(\PageIndex{20}\).

Figure \(\PageIndex{20}\): Summary of transaldolase reaction

As with transketolase, the transaldolase enzyme is reversible. Again, note the number of carbons in the reactants and products: 3C + 7C ↔ 4C + 6C. Unlike the transketolase used in the last reaction, the transaldolase enzyme does NOT use TPP as a cofactor. Instead, it forms a Schiff base intermediate similar to the aldolase enzyme in the glycolytic pathway. The enzyme removes a 3C ketol group (dihydroxyacetone) from sedoheptulose 7-phosphate and transfers it to glyceraldehyde 3-phosphate, forming fructose 6-phosphate. Erythrose 4-phosphate is left from the original sedoheptulose 7-phosphate.

We will explore the mechanism of the E. coli enzyme in reverse. The first part of the mechanism of transaldolase is shown in Figure \(\PageIndex{21}\).

Figure \(\PageIndex{21}\): Mechanism for the first half of the transaldolase reaction. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/148/

In the first part of the reaction, fructose 6-phosphate binds to the enzyme's active site, where an active site lysine residue mediates nucleophilic attack on the carbonyl carbon and forms a covalent intermediate with the enzyme. Formation of the Schiff base leads to dehydration of the intermediate. The Schiff base nitrogen becomes protonated, leading to the oxidation of the C4 hydroxyl group and subsequent bond cleavage, releasing glyceraldehyde 3-phosphate. The remaining ES-complex rearranges to form an enol intermediate.

Figure \(\PageIndex{22}\) shows the mechanism for the second half of the transaldolase reaction.

Figure \(\PageIndex{22}\): Mechanism for the second half of the transaldolase reaction. https://www.ebi.ac.uk/thornton-srv/m-csa/entry/148/

Once glyceraldehyde 3-phosphate has left the enzyme's active site, erythrose 4-phosphate can bind. The enol from the ES intermediate mediates nucleophilic attack on the aldehyde carbonyl group of erythrose 4-phosphate. This results in the formation of a Schiff base intermediate. Hydration at the Schiff base carbon atom ensues, followed by the oxidation of the newly incorporated alcohol to form a ketone functional group. The formation of the ketone causes bond cleavage between the enzyme and the newly formed ketose, sedoheptulose 7-phosphate.

Figure \(\PageIndex{23}\) shows an interactive iCn3D model of transaldolase B from Escherichia coli (1ONR).

Figure \(\PageIndex{23}\): 3D structure of Transaldolase B from E. coli (1ONR).  (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...7swLZVpNPe3eU9

Transketolase

As seen above, fructose 6-phosphate is a product of the transaldolase enzyme during the nonoxidative branch of the PPP. This sugar can feed back into the glycolytic cycle. The formation of fructose 6-phosphate can also occur using the transketolase enzyme when it transfers a 2C ketol group from xylulose 5-phosphate to the aldose, erythrose 4-phosphate. This reaction is summarized in Figure \(\PageIndex{24}\).

Figure \(\PageIndex{24}\) Summary of the second transketolase reaction during the pentose phosphate pathway.

Both products of this final transketolase reaction can be used for energy production via glycolysis. Thus, there are clear metabolic ties between the PPP and the energy-producing glycolytic pathway, and intermediates from both pathways can readily be exchanged through the activity of these reversible enzymes. The PPP provides key intermediates, including ribose 5-phosphate, which is used in the biosynthesis of nucleotides and, ultimately, nucleic acids, as well as other important metabolic molecules such as FAD, NAD⁺, NADP⁺, and Coenzyme A. In addition to being a biosynthetic source for these molecules, the PPP is also the primary source for reducing NADP⁺ to NADPH during the oxidative reactions. NADPH is used as an electron donor in many biosynthetic processes. For example, the biosynthesis of many lipid molecules requires NADPH, including the production of triacylglycerols, phospholipids, and steroids. NADPH is also required for the biosynthesis of some amino acids (such as glutamate and proline) and for reducing ribonucleotides and deoxyribonucleotides during the synthesis of RNA and DNA. NADPH is also utilized by many oxidoreductases involved in detoxification reactions within the body. Within the immune system, NADPH oxidases, or NOX enzymes, produce superoxide and are used to nonspecifically damage invading pathogens. NADP⁺/NADPH ratios can also play a regulatory role in cellular metabolic processes and are used as allosteric effectors for several enzymes and oxidation-sensing proteins.

Regulation of the Pentose Phosphate Pathway

The cellular demand for the two major products of the PPP (ribose 5-phosphate and reduced NADPH) can differ depending on the cell type or the current cellular environment, such as increased metabolic demand or oxidative stress. Thus, the two major products may need to be produced independently and in different quantities. For example, we can imagine situations in which the demand for ribose-5-phosphate and NADPH is balanced with the standard PPP reactions. However, we can also imagine times when the demand for ribose 5-phosphate may be much higher than for NADPH, or vice versa, when the demand for NADPH may be much greater than for ribose 5-phosphate. Thus, regulatory strategies enable these different pools to be produced independently of one another and to adapt to cellular needs. This ability largely depends on the production of metabolic intermediates that can be readily interchanged within the glycolytic pathway.

For example, the NADP⁺/NADPH ratio serves as a key regulator of the PPP's oxidative branch. The first enzymatic step of the pathway mediated by the glucose 6-phosphate dehydrogenase (G6PD) is regulated in this fashion. It helps control the pool of glucose-6-phosphate used in the PPP to produce reduced NADPH. Low levels of NADP⁺ inhibit the G6PD enzyme. The G6PD reaction is essentially irreversible and serves as the committed step for glucose to enter the PPP's oxidative part. Thus, regulating this enzymatic step is key to controlling NADPH levels within the cell. When the ratio of NADP⁺/NADPH increases, G6PD becomes more active, and the reduction of NADP⁺ to NADPH increases.

When ribose 5-phosphate is in high demand, its cellular concentration decreases, driving the ribose 5-phosphate isomerase reaction forward by mass action. This enzyme will then convert ribulose 5-phosphate, generated in the oxidative branch of the pentose phosphate pathway, into ribose 5-phosphate to replenish the pool and meet biosynthetic needs. However, suppose cellular demand for ribose 5-phosphate exceeds the amount generated by the oxidative branch. In that case, additional ribose 5-phosphate can be produced in the nonoxidative branch of the pentose phosphate pathway through the rearrangement of glycolytic intermediates.

The opposite also occurs when there is a high need for NADPH in the cell but a low need for ribose 5-phosphate. In this situation, ribose-5-phosphate is converted to fructose and glyceraldehyde-3-phosphate, which can be further metabolized in the glycolytic pathway.

In addition, PPP activity varies depending on tissue type and body location. For example, skeletal muscle has very low PPP activity, as this tissue requires greater energy production and greater glycolytic pathway activity. On the other hand, the PPP is highly active in adipose tissue due to the heightened requirement for intermediates needed for lipid biosynthesis.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter describes the pentose phosphate pathway (PPP), a cytosolic branch of glucose metabolism that diverges from glycolysis after the formation of glucose-6-phosphate and serves two principal biosynthetic functions: generation of NADPH for reductive biosynthesis and antioxidant defense, and generation of ribose-5-phosphate for nucleotide and nucleic acid synthesis. Unlike glycolysis, the PPP produces no ATP; its value lies entirely in the metabolic currency of its two products.

The pathway consists of two distinct branches. The oxidative branch is irreversible and produces two molecules of NADPH per glucose-6-phosphate consumed through three sequential reactions. Glucose-6-phosphate dehydrogenase (G6PD) catalyzes the first and committed step — oxidation of glucose-6-phosphate to 6-phosphogluconolactone — and is the major regulatory enzyme of the branch. Its activity is controlled by the NADP⁺/NADPH ratio: when NADPH is consumed, and NADP⁺ accumulates, G6PD is activated, increasing flux through the oxidative branch. 6-Phosphogluconolactonase then hydrolyzes the lactone to 6-phosphogluconate via a proton-relay mechanism involving conserved histidine and aspartate residues. Finally, 6-phosphogluconate dehydrogenase catalyzes an oxidative decarboxylation, proceeding through a keto intermediate and an enediol, to yield ribulose-5-phosphate and a second NADPH.

The NADPH produced by the oxidative branch is critical for maintaining the reduced glutathione (GSH) pool via glutathione reductase. GSH, in turn, serves as a substrate for glutathione peroxidase and catalase, which neutralize hydrogen peroxide and other ROS. This function is especially important in erythrocytes, which lack mitochondria and therefore depend exclusively on the PPP for cytosolic NADPH. G6PD deficiency — one of the most common enzyme deficiencies worldwide, with a geographic distribution mirroring malaria endemicity — impairs this protective capacity, rendering red blood cells vulnerable to oxidative lysis and causing hemolytic anemia upon exposure to oxidative triggers such as infection or fava beans. Many disease-causing mutations cluster near the structural NADP⁺ binding site, destabilizing the enzyme without directly abolishing its active site.

The non-oxidative branch consists of a series of fully reversible sugar interconversion reactions that connect the PPP to glycolysis and gluconeogenesis. Ribose-5-phosphate isomerase and ribulose-5-phosphate epimerase convert ribulose-5-phosphate to ribose-5-phosphate and xylulose-5-phosphate, respectively, both proceeding through enediol intermediates. Transketolase, a TPP-dependent homodimer, then transfers a two-carbon ketol unit from xylulose-5-phosphate to ribose-5-phosphate, yielding glyceraldehyde-3-phosphate (3C) and sedoheptulose-7-phosphate (7C). The TPP cofactor forms a covalent adduct with the donor ketose, stabilizing the transferred fragment as a carbanion intermediate. Transaldolase subsequently transfers a three-carbon dihydroxyacetone unit from sedoheptulose-7-phosphate to glyceraldehyde-3-phosphate via a covalent Schiff base intermediate with an active-site lysine, producing erythrose-4-phosphate (4C) and fructose-6-phosphate (6C). A second transketolase reaction then converts xylulose-5-phosphate and erythrose-4-phosphate to fructose-6-phosphate and glyceraldehyde-3-phosphate, both of which re-enter glycolysis. The carbon-counting logic of these reactions — 5+5→3+7, 3+7→4+6, 5+4→3+6 — reflects the elegant rearrangement of carbon skeletons that allows the cell to interconvert pentose phosphates and glycolytic intermediates bidirectionally.

Regulation of the PPP allows the cell to independently tune NADPH and ribose-5-phosphate production to meet changing demands. When ribose-5-phosphate demand is high, mass action drives the isomerase reaction forward; if demand exceeds the oxidative branch output, the non-oxidative branch can run in reverse, drawing glycolytic intermediates into ribose-5-phosphate synthesis. Conversely, when NADPH demand is high but ribose-5-phosphate is not needed, the non-oxidative branch converts pentose phosphates back to fructose-6-phosphate and glyceraldehyde-3-phosphate, allowing the oxidative branch to continue running without net accumulation of pentose phosphates. Tissue-specific variation in PPP activity reflects these priorities: skeletal muscle, with its emphasis on ATP production, has low PPP flux, while adipose tissue, liver, and adrenal cortex — all active in reductive biosynthesis of fatty acids and steroids — have high PPP activity.

References:

Edwards et al.: Structural characterization of a ribose5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis. BMC Structural Biology 2011 11:39. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3212906/

Ge T, Yang J, Zhou S, Wang Y, Li Y and Tong X (2020) The Role of the Pentose Phosphate Pathway in Diabetes and Cancer. Front. Endocrinol. 11:365. doi: 10.3389/fendo.2020.00365

Polat, I.H., Tarrado-Castellarnau, M., Bharat, R., Perarnau, J., Benito, A., Cortes, R., Sabatier, P., and Cascante, M. (2021) Oxidative Pentose Phosphate Pathway Enzyme 6-Phosphogluconate Dehydrogenase Plays a Key Role in Breast Cancer Metabolism. Biology 10(2):85. https://doi.org/10.3390/biology10020085

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