7 Pentose Phosphate Pathway (PPP), Purine and Pyrimidine Metabolism

Learning Objectives

  • Describe the role of NADPH produced by the pentose phosphate pathway in metabolism and regulation of glucose 6-phosphate dehydrogenase.
  • Determine the utility of the oxidative and nonoxidative portions of the pentose phosphate pathway (PPP) and how these pathways interface with glycolysis.
  • Describe the amino acid composition of glutathione (GSH) and understand the role of GSH in attenuating oxidative damage.
  • Describe how the pentose phosphate pathway and the process of DNA replication interface with the biosynthesis of purine and pyrimidine nucleotides.
  • Evaluate the central role of 5-phosphoribosyl-1-pyrophosphate (PRPP) in nucleotide metabolism.
  • Describe the purine salvage pathway, specifically the reaction catalyzed by hypoxanthine-guanine phosphoribosyltransferase (HGPRT).
  • Identify the key regulatory steps in both purine and pyrimidine synthesis, and evaluate flux through each pathway depending on levels of allosteric activators and inhibitors.
  • Describe conditions that lead to elevated orotic acid, and interpret urine orotic acid concentration for the diagnosis of defects of the urea cycle or pyrimidine biosynthesis.

About this Chapter

As we have seen previously, glucose can be diverted to several different pathways depending on metabolic needs. One of these pathways is the pentose phosphate pathway, which plays an integral role in producing both NADPH and the five-carbon sugar ribose. NADPH provides the cell with an energy source for reductive biosynthesis and detoxification of free radicals, while ribose is an essential component in the synthesis of both purine and pyrimidine nucleotides. Aberrations (increases or decreases) in either of these metabolic pathways, PPP or nucleotide synthesis, can result in the common clinical presentations of anemia, jaundice, or gout.

7.1 Pentose Phosphate Pathway

The pentose phosphate pathway (PPP — also known as the hexose monosphosphate shunt) is a cytosolic pathway that interfaces with glycolysis. In this pathway, no ATP is directly produced from the oxidation of glucose 6-phosphate; instead the oxidative portion of the PPP is coupled to the production of NADPH. In addition to generating NADPH, which is essential for detoxification reactions and fatty acid synthesis, it also produces five-carbon sugars required for nucleotide synthesis.

Oxidative and nonoxidative functions

There are two parts of the pathway that are distinct and can be regulated independently. The first phase, or oxidative phase, consists of two irreversible oxidations that produce NADPH. As noted above, NADPH is required for reductive detoxification and fatty acid synthesis. (NADPH is not oxidized in the ETC.) In the red blood cell, this is extremely important as the PPP pathway provides the only source of NADPH. NADPH is essential to maintain sufficient levels of reduced glutathione in the red blood cell. Glutathione is a tripeptide commonly used in tissues to detoxify free radicals and reduce cellular oxidation.

The nonoxidative phase of the pathway allows for the conversion of ribulose 5-phosphate into ribose 5-phosphate, which is needed for nucleotide synthesis (figure 7.1). All of these interconversions in the nonoxidative pathway are reversible and use the enzymes transketolase or transaldolase to move two-carbon or three-carbon units on to other sugar moieties to generate a variety of sugar intermediates. Transketolase requires thiamine pyrophosphate (TPP) as a cofactor. This is of clinical relevance as TPP levels can be measured by addressing the activity of transketolase in a blood sample. A reduction in transketolase activity is an indicator of a thiamine deficiency.

Glycolysis: Glucose arrow Glucose 6-phosphate arrow Fructose 6-phosphate arrow glyceraldehyde 3-phosphate arrow with loss of NADH and ATP to pyruvate. Pentose phosphate pathway: Glucose 6-phosphate arrow with text oxidative to ribulose 5-phosphate bidirectional arrow xylulose 5-phosphate bidirectional arrow with text non-oxidative fructose 6-phosphate. On oxidative arrow, CO2 leaves and 2 NADP+ arrow 2 NADPH arrows to fatty acid synthesis, glutathione reduction, and other reactions (such as detoxification). On non-oxidative arrow, arrows to Ribose 5-phosphate and glyceraldehyde 3-phosphate. Ribulose 5-phosphate bidirectional arrow ribose 5-phosphate arrow nucleotide biosynthesis
Figure 7.1: Overview of the pentose phosphate pathway and its interface with glycolysis.

Any compounds unused by the nonoxidative pathway will eventually be converted to fructose 6-phosphate or glyceraldehyde 3-phosphate, both of which will re-enter the glycolytic pathway (figures 7.1 and 7.2).

Glyceraldehyde-3-phosphate bidirectional arrow Fructose-1,6-biphosphate forward arrow with enzyme fructose-1,6-biphosphatase and loss of Pi to Fructose-6-phosphate. Fructose-6-phosphate backwards arrow with ATP arrow ADP to Fructose-1,6-biphosphate. Fructose6-phosphate bidirectional arrow with enzyme phosphoglucose isomerase to Glucose-6-phosphate bidirectional arrow with enzyme Glucose-6-phosphate dehydrogenase (G6PD) and biosynthetic reduction reactions of NADPH bidirectional arrow NADP+ and NADPH arrow NADP+, glutathione reductase of glutathione (oxidized) arrow glutathione (reduced) touching previous arrow, and arrow glutathione (oxidized), glutathione peroxidase of H2O2 arrow 2 H2O touching the previous arrow to 6-phosphogluconolactone arrow with enzyme lactonase and H2O arrow H+ to 6-phosphogluconate arrow with enzyme 6-phosphogluconate dehydrogenase, NADP+ arrow NADPH, and loss of CO2 to ribulose 5-phosphate. Ribulose 5-phosphate bidirectional arrow ribose 5-phosphate isomerase to ribose 5-phosphate arrow with enzyme PRPP synthetase and ATP arrow ADP to 5-phosphoribosyl pyrophosphate (PRPP) arrow nucleotides. Ribulose 5-phosphate bidirectional arrow with enzyme Ribulose 5-phosphate epimerase to xylulose 5-phosphate bidirectional arrow with enzyme transketolase and 5+3 to ribose 5-phosphate. Glyceraldehyde 3-phosphate bidirectional arrow touching previous arrow with 7+3 to sedoheptulose 7-phosphate bidirectional arrow with enzyme transaldolase and 7+3 to glyceraldehyde 3-phosphate. Erythrose 4-phosphate bidirectional arrow with 6+4 touching the previous arrow to Fructose 6-phosphate. Glyceraldehyde 3-phosphate bidirectional arrow with enzyme transketolase and 6+3 to fructose 6-phosphate. Xylulose 5-phosphate bidirectional arrow touching previous arrow with 4+5 to erythrose 4-phosphate.
Figure 7.2: Pentose phosphate pathway and its connection to glycolysis and glutathione synthesis.

Regulation of the pentose phosphate pathway

The key regulatory enzyme for the pentose phosphate pathway is within the oxidative portion. Glucose 6-phosphate dehydrogenase oxidizes glucose 6-phosphate to 6-phosphogluconolactone, and is regulated by negative feedback. In this two-step reaction NADPH is also produced, and high levels of NADPH will inhibit the activity of glucose 6-phosphate dehydrogenase. This ensures NADPH is only generated as needed by the cell; this is the primary regulatory mechanism within the pathway.

The nonoxidative phase is not regulated; however, in conditions where there is a high demand for nucleotide production (such as in the case for highly proliferative cells), the nonoxidative part of the pathway can function independently of the oxidative phase to produce ribose 5-phosphate from the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate (figure 7.2).

 

 

Requirement of the pentose phosphate pathway in RBCs

The two essential products of this pathway are NADPH and ribose 5-phosphate. NADPH is a high-energy compound often used for reductive biosynthesis as it cannot be oxidized in the ETC. It is also used by many tissues to scavenge (and detoxify) reactive oxygen species (ROS) before causing cellular damage. This is especially important in red blood cells; RBCs lack malic enzyme, making this the only pathway that can generate NADPH. A lack of NADPH in RBCs (such as due to a glucose 6-phosphate dehydrogenase deficiency) can cause excessive hemolysis, leading to the clinical presentation of jaundice (figure 7.3).

Glucose arrow into an erythrocyte to glucose 6-phosphate. Glucose 6-phosphate arrow glycolysis arrow with loss of 2 ATP and NADH to 2 pyruvate arrow 2 lactate (pentose phosphate pathway). Glucose 6-phosphate arrow with enzyme glucose 6-phosphate dehydrogenase to 6-phosphogluconate arrow to glycolysis. 3 clockwise circular arrows touching the arrow between glucose 6-phosphate to 6-phosphogluconate. NADP+ counterclockwise circular arrow with enzyme glucose 6-phosphate dehydrogenase to NADPH + H+ counterclockwise circular arrow with enzyme glutathione reductase to NADP+. Horizontal line touching arrows with glucose 6-phosphate dehydrogenase labeled glucose 6-phosphate dehydrogenase deficiency. 2 GSH clockwise circular arrow with enzyme glutathione reductase to GS-SG clockwise circular arrow with enzyme glutathione peroxidase to 2 GSH. GS-SG arrow Heinz bodies. ROS circular arrow to H2O2 counterclockwise circular arrow with enzyme glutathione peroxidase to 2 H2O. Oxidant stress: infection, certain drugs, fava beans arrows to H2O2 and HO radical. HO radical arrow hemolysis. Below circular arrow Oxy Hb arrows to O2- and MetHb. MetHb arrow Heinz bodies
Figure 7.3: NADPH in the red blood cell as a means of reducing glutathione.

Glutathione (GSH) is a tripeptide compound consisting of glutamate, cysteine, and glycine. It plays a key role in scavenging reactive oxygen species (ROS), which cause both DNA and cellular/protein damage. Reduction of GSH in the red blood cell is done exclusively through a series of oxidation reduction reactions using NADPH. The loss of NADPH in RBCs therefore increases ROS and can lead to hemolysis (figure 7.3).

Summary of pathway regulation

Metabolic pathway Major regulatory enzyme Allosteric effectors Hormonal effects
Pentose phosphate pathway Glucose 6-phosphate dehydrogenase NADPH (-) None

Table 7.1: Summary of pathway regulation.

7.1 References and resources

Text

Ferrier, D. R., ed. Lippincott Illustrated Reviews: Biochemistry, 7th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, 2017, Chapter 13: Pentose Phosphate Pathway and NAPDH, Chapter 22: Nucleotide Metabolism.

Le, T., and V. Bhushan. First Aid for the USMLE Step 1, 29th ed. New York: McGraw Hill Education, 2018, 35–37, 79.

Lieberman, M., and A. Peet, eds. Marks’ Basic Medical Biochemistry: A Clinical Approach, 5th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, 2018, Chapter 27: Pentose Phosphate Pathway, Chapter 39: Purine and Pyrimidine Synthesis.

Figures

Grey, Kindred, Figure 7.2 Pentose pathway and its connection to glycolysis and glutathione synthesis. 2021. https://archive.org/details/7.2_20210926. CC BY 4.0.

Lieberman M, Peet A. Figure 7.1 Overview of the pentose phosphate pathway and its interface with glycolysis. Adapted under Fair Use from Marks’ Basic Medical Biochemistry. 5th Ed. pp 543. Figure 27.1 Overview of the pentose phosphate pathway. 2017.

Lieberman M, Peet A. Figure 7.3 NADPH in the red blood cell as a means of reducing glutathione. Adapted under Fair Use from Marks’ Basic Medical Biochemistry. 5th Ed. pp 549. Figure 27.7 Hemolysis caused by reactive oxygen species (ROS). 2017.

7.2 Nucleotide Synthesis

Nucleotides are the fundamental building blocks essential for the synthesis of DNA and RNA. Each nucleotide contains three functional groups: a sugar, a base, and phosphate (figure 7.4).

Pentagon shaped 5-carbon sugar with oxygen at top, position 1 carbon and base, position 2 carbon and OH group, position 3 carbon and OH group, position 4 carbon attached to 3 phosphate groups. Nucleoside contains the sugar and base. Nucleoside monophosphate (NMP) contains the sugar, base, and one phosphate group. Nucleoside diphosphate (NDP) contains the sugar, base, and two phosphate groups. Nucleoside triphosphate (NTP) contains the sugar, base, and three phosphate groups. NMP, NDP, and NTP are nucleotides.
Figure 7.4: Basic structure of nucleotides.

Nucleotides can be divided into two groups: pyrimidines and purines. The family of pyrimidines includes thymine (T), cytosine (C), and uracil (U), which is only incorporated into RNA. These compounds contain a single-ringed nitrogenous base that pairs with a purine nucleotide counterpart. Thymine pairs with adenine forming two hydrogen bonds, in contrast to cytosine, which pairs with guanine to form three hydrogen bonds. Purines, both guanine (G) and adenine (A), are double-ringed structures and more difficult to break down in the body. As such, the salvage pathway for purine metabolism is of importance (figure 7.5).

Cytosine IUPAC ID: 6-amino-1H-pyrimidin-2-one. Thymine IUPAC ID: 5-methyl-1H-pyrimidine-2,4-dione. Guanine IUPAC ID: 2-amino-1,7-dihydropurin-6-one. Adenine IUPAC ID: 7H-purin-6-amine.
Figure 7.5: Overview of purine and pyrimidine bases.

Nucleotide synthesis will be described below, but one of the fundamental requirements of the synthesis of either purines or pyrimidines is the need for a five-carbon sugar (ribose). This sugar is generated through glucose oxidation via the pentose phosphate pathway.

For purines synthesis, the base is synthesized and attached to the sugar, while for pyrimidine synthesis, the sugar group is added after the base is produced. In either case, ribose is the added sugar, and this must be converted to the deoxyribose form before the bases can be used for DNA synthesis.

Conversion of ribose to deoxyribose nucleotides

All bases are synthesized in the ribose form and used directly for transcription. They can be converted to the deoxy form, which is needed for DNA replication. The enzyme, ribonucleotide reductase, converts the diphosphate form of a ribose base to the deoxybase form. The enzyme has two sites for regulation: an enzyme activity site and a substrate specificity site. The enzyme activity site must have ATP/ADP bound for the enzyme to be active, while the substrate specificity site will bind different nucleotides influencing the enzyme substrate preference, therefore altering which base is being acted upon depending on cellular needs.

Generation of 5-phosphoribosyl-1-phosphate (PRPP)

Ribose 5-phosphate is not used directly for either purine or pyrimidine synthesis, rather it is used to synthesize the “active pentose” — 5-phosphoribosyl-1-pyrophosphate (PRPP). The conversion is catalyzed by the enzyme phosphoribosyl-1-pyrophosphate (PRPP) synthase. PRPP is the activated five-carbon sugar used for nucleotide synthesis and provides both the sugar and phosphate group to nucleotides (figure 7.6).

Ribose 1-P bidirectional arrow with enzyme phosphopentomutase to ribose 5-P arrow with ATP arrow AMP and enzyme PRPP synthetase to PRPP. Pi excites and purine bases inhibits PRPP synthetase
Figure 7.6: Synthesis of PRPP and regulation of PRPP synthetase.

Regulation of PRPP synthase

The enzyme, PRPP synthetase, is activated by Pi (inorganic phosphate) and inhibited by the purine bases adenine and guanine.

Synthesis of purines

Purines are composed of a bicyclic structure that is synthesized from carbon and nitrogen donated from various compounds such as carbon dioxide, glycine, glutamine, aspartate, and tetrahydrofolate (TH4). The synthesis of purines starts with the synthesis of 5ʼphosphoribosylamine from PRPP and glutamine. The enzyme glutamine phosphoribosylpyrophate amidotransferase (GPAT) catalyzes this reaction and is the committed step in purine synthesis (figure 7.7). Synthesis continues for nine additional steps culminating in the synthesis of inosine monophosphate (IMP), which contains the base hypoxanthine. IMP is used to generate both AMP and GMP. The synthesis of both AMP and GMP requires energy in the form of the alternative base (i.e., the synthesis of GMP requires ATP while AMP synthesis requires energy in the form of GTP). The synthesis of AMP and GMP is regulated by feedback inhibition (figures 7.7 and 7.8). This allows for the maintenance of nucleotides in a relative ratio that is required for cellular processes. The generated nucleotide monophosphates can be converted to the di and triphosphate forms by nucleotide specific kinases, which will transfer phosphate groups to maintain a balance of the mono, di, and triphosphate forms.

5-phosphoribosyl pyrophosphate (PRPP) arrow enzyme glutamine PRPP amidotransferase (GPAT) with Glutamine arrow Glutamate and loss of PPi to 5-phosphoribosylamine arrow enzyme GAR synthetase and Glycine addition, ATP arrow ADP, loss of Pi to glycinamide ribonucleotide (GAR) arrow enzyme GAR transformylase and N10-formyl-THF arrow THF to formyl-GAR (FGAR) arrow enzyme FGAM synthetase and Glutamine arrow Glutamate, ATP arrow ADP, loss of Pi and H2O to formiminoglycinamide ribonucleotide (FGAM) arrow enzyme AIR synthetase with ATP arrow ADP, loss of Pi to aminoimidazole ribonucleotide (AIR) arrow enzyme AIR carboxylase with CO2 addition to carboxyaminoimidazole ribonucleotide (CAIR) arrow enzyme SAICAR synthetase and aspartate addition, ATP arrow ADP, and loss of Pi to succinylaminoimidazole carboxyamide ribonucleotide (SAICAR) arrow enzymeadenylosuccinate lyase with H2O addition and loss of fumarate to aminoiminidazole carboxamide ribonucleotide (AICAR) bidirectional arrow enzyme AICAR transformylase with N10-formyl-THF bidirectional arrow THF to formaminoimidazole carboxyamide ribonucleotide (FAICAR) arrow enzyme IMP cyclohydrolase and loss of H20 to inosine monophosphate (IMP)
Figure 7.7: Overview of purine synthesis. The reaction catalyzed by GPAT is the regulatory enzyme of the pathway.

 

Ribose-5-phosphate arrow enzyme PRPP synthetase to 5-phosphoribosyl-1-pyrophosphate (PRPP) arrow enzyme glutamine phosphoribosyl amidotransferase to 5-phosphoribosyl-1-amine arrow IMP arrow enzyme IMP dehydrogenase to XMP arrow GMP arrow GDP arrow GTP. IMP arrow enzyme adenylosuccinate synthetase to adenylosuccinate arrow AMP arrow ADP arrow ATP. GDP and ADP inhibit PRPP synthetase. GMP, GTP, AMP, and ATP inhibit glutamine phosphoribosyl amidotransferase. GMP inhibits IMP dehydrogenase. AMP inhibits adenylosuccinate synthetase.
Figure 7.8: Purine synthesis and regulation of glutamine: phosphoribosylpyrophosphate amidotransferase.

Regulation of purine synthesis

The regulatory enzyme GPAT is allosterically activated by PRPP and inhibited by IMP, AMP, and GMP. All three must be present to inhibit activity of this enzyme.

Degradation of purines

Like amino acids, nucleotides contain nitrogen and must be degraded in a manner that allows for proper nitrogen disposal either through the urea cycle or by the synthesis of a nontoxic compound.

Degradation of dietary nucleotides occurs in the gut, while nucleotides from de novo synthesis are degraded in the liver. The fundamental process involves the dismantling of the sugar, phosphate, and base structure into their own respective units (figure 7.9). In the case of purine degradation, the base is excreted in the form of uric acid. Purine nucleoside phosphorylase converts inosine and guanosine to their respective bases (hypoxanthine and guanine). Finally, xanthine oxidase will oxidize hypoxanthine to xanthine (guanine can be deaminated to xanthine), and xanthine can be further oxidized to uric acid by the same enzyme. Uric acid is excreted in the urine.

RNA/DNA arrow (deoxy) GMP arrow enzyme nucleotidase with H2O arrow Pi to (deoxy) guanosine arrow enzyme purine nucleoside phosphorylase and Pi bidirectional arrow (deoxy) ribose-1-P to guanine arrow enzyme guanine deaminase and H2O arrow NH4+ to xanthine arrow enzyme xanthine oxidase and O2 addition and H2O arrow H2O2 to uric acid. RNA/DNA arrow (deoxy) AMP arrow enzyme nucleotidase with H2O arrow Pi to (deoxy) adenosine arrow enzyme adenosine deaminase with H2O arrow NH4+ to (deoxy) inosine. (deoxy) AMP arrow enzyme AMP deaminase with H2O arrow NH4+ to (deoxy) IMP arrow enzyme nucleotidase with H2O arrow Pi to (deoxy) inosine bidirectional arrow enzyme purine nucleoside phosphorylase with Pi bidirectional arrow (deoxy) ribose-1-P to hypoxanthine arrow enzyme xanthine oxidase with H2O addition and O2 arrow H2O2 to xanthine. RNA/DNA arrow (deoxy) CMP arrow enzyme nucleotidase and H2O arrow Pi to(deoxy) cytidine arrow enzyme pyrimidine nucleoside deaminase with loss of NH4+ to (deoxy) uridine. RNA/DNA arrow UMP arrow enzyme nucleotidase with H2O arrow Pi to (deoxy) uridine bidirectional arrow enzyme uridine phosphorylase and Pi bidirectional arrow (deoxy) ribose-1-P to uracil arrow enzyme dihydropyrimidine dehydrogenase (DPD) with NADPH arrow NADP+ to dihydrouracil arrow enzyme dihydropyrimidinase to β-ureidopropionate arrow enzyme ureidopropionase with loss of CO2 and NH4+ to β-alanine. RNA/DNA arrow (deoxy) TMP arrow enzyme nucleotidase with H2O arrow Pi to thymidine bidirectional arrow enzyme thymidine phosphorylase with Pi bidirectional arrow (deoxy) ribose-1-P to thymine arrow with NADPH arrow NADP+ to dihydrothymine arrow β-ureidoisobutyrate arrow with loss of CO2 and NH4+ to β-aminoisobutyric acid.
Figure 7.9: Breakdown of nucleotides.

Excess uric acid, hyperuricemia, can cause the precipitation of uric acid crystals in the joints eliciting an inflammatory reaction causing acute pain or gout. The majority of individuals diagnosed with gout present due to underexcretion of uric acid. And this can be caused by the presence of other pathologies, such as lactic acidosis or the use of diuretics. Less common presentations of gout are associated with overproduction of uric acid, which can be caused by increased activity of PRPP synthetase or deficiency in purine recycling enzyme HGPRT caused by Lesch-Nyhan syndrome (figure 7.10).

Hypoxanthine arrow enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) with PRPP arrow PPi to inosine monophosphate (IMP) arrow enzyme IMP dehydrogenase with H2O addition and NAD+ arrow NADH to XMP arrow enzyme GMP synthetase with Glutamine arrow Glutamate and ATP arrow AMP + PPi to GMP arrow enzyme GMP kinase with ATP arrow ADP to GDP arrow enzyme nucleoside diphosphate kinase with ATP arrow ADP to GTP arrow biopterin. IMP arrow enzyme adenylosuccinate synthetase with GTP arrow GDP, aspartate addition and loss of Pi to adenylosuccinate arrow enzyme adenylosuccinate lyase with loss of fumarate to AMP arrow enzyme adenylate kinase with ATP arrow ADP to ADP arrow enzyme nucleotide diphosphate kinase with GTP arrow GDP to ATP arrow S-adenosylmethionine, coenzyme A, NADH, FADH2. Adenine arrow enzyme adenine phosphoribosyltransferase with PRPP arrow Pi to AMP arrow enzyme AMP deaminase with loss of NH4+ to IMP. Uridine bidirectional arrow enzyme uridine phosphorylase with loss of uracil and Pi to ribose-1-P bidirectional arrow enzyme phosphomannomutase to Ribose-5-P arrow enzyme PRPP synthetase with ATP arrow AMP to PRPP. Guanine arrow enzyme HGPRT with PRPP arrow PPi to GMP arrow enzyme GMP reductase with NADPH arrow NADP+ and loss of NH4+ to IMP.
Figure 7.10: Nucleotide base salvage. Reaction catalyzed by HGPRT is clinically relevant as deficiencies can cause accumulation of uric acid.

Secondary hyperuricemia is also seen in individuals with myeloproliferative disorders undergoing therapy where there is excess cellular turnover (cell lysis leads to an accumulation of nucleotides) or in cases of Von Gierke disease or fructose intolerance, which increases substrate for PRPP synthesis. Xanthine oxidase inhibitors, such as allopurinol, are used as part of the management of gout.

Salvage of purines

The ability to recycle nucleotides is specifically important in the case of purines as de novo synthesis uses much more ATP than salvage. The degradation product of purine bases is uric acid, which is an insoluble compound, and accumulation can result in several clinical disorders as previously discussed. As such, purine bases can also undergo salvage reaction where bases are recycled and used in a new process. To reduce the amount of uric acid production, purines can be salvaged and reconverted back to their triphosphate form to be reused. There are two primary enzymes involved in the salvage pathway: adenine phosphoribosyltransferase (APRT) and xanthine-guanine phosphoribosyltransferase (HGPRT) (figure 7.10). These enzymes will recombine the base (either adenine, guanine, or hypoxanthine) with PRPP to generate AMP, GMP, or IMP respectively. Adenosine is the only nucleoside that can be rephosphorylated to its monosphosphate form using adenosine kinase (figure 7.11). All other nucleosides must be degraded to their free base before they can be salvaged.

Adenosine arrow with ATP arrow ADP and enzyme adenosine kinase to adenosine 5’-monophosphate. Cytidine arrow with ATP arrow ADP and enzyme uridine-cytidine kinase to cytidine monophosphate. Uridine arrow with ATP arrow ADP with enzyme uridine-cytidine kinase to uridine 5’-monophosphate. Deoxycytidine arrow with ATP arrow ADP and enzyme deoxycytidine kinase to deoxycytidine 5’-monophosphate. Thymidine arrow with ATP arrow ADP and enzyme thymidine kinase to deoxythymidine 5’ monophosphate. Deoxyuridine arrow with ATP arrow ADP and enzyme thymidine kinase to deoxyuridine 5’-monophosphate.
Figure 7.11: Nucleotide specific pathways for base salvage.

Synthesis of pyrimidines

HCO3- arrow enzyme carbamoyl phosphate synthetase II (CPSII) with 2 ATP addition, glutamine arrow glutamate, loss of 2 ADP and Pi to carbamoyl phosphate arrow enzyme aspartate transcarbamoylase with aspartate addition and loss of Pi to N-carbamoyl aspartate arrow enzyme dihydroorotase to dihydroorotate arrow enzyme dihydroorotate dehydrogenase (in mitochondria) with NAD+ arrow NADH to orotate arrow enzyme orotate phosphoribosyltransferase with PRPP arrow PPi to orotidine-5’-monophosphate (OMP) arrow enzyme OMP decarboxylase (UMP synthase) with loss of CO2 to uridine-5’-monophosphate (UMP) arrow enzyme UMP kinase with ATP arrow ADP to UDP arrow enzyme nucleoside diphosphate kinase with ATP arrow ADP to UTP arrow enzyme CTP synthetase with ATP arrow ADP + Pi and glutamine arrow glutamate to CTP arrow UTP with NH4+ leaving (spontaneous). PRPP and ATP excite, and UTP inhibits CPSII.
Figure 7.12: Overview of pyrimidine synthesis. The reaction catalyzed by carbamoyl phosphate synthetase I is the regulatory enzyme of the pathway.

In contrast to purine synthesis, the pyrimidine bases are synthesized before the ribose sugar and phosphate groups are added in the form of PRPP (figure 7.12). The initial step of the pathways involves the synthesis of carbamoyl phosphate from glutamine, carbon dioxide, and 2 ATP. Carbamoyl phosphate synthetase II (CSPII) catalyzes this reaction. (Note there is an analogous enzyme in the mitochondria for the urea cycle termed carbamoyl phosphate synthetase I, which also generates carbamoyl phosphate.) Of clinical importance is the intermediate orotate. Elevations of orotate (orotic acid) are consistent with enzymatic deficiencies in this pathway or urea cycle deficiencies such as a defect in ornithine transcarbamoylase. In the case of a urea cycle deficiency, an excess carbamoyl phosphate can enter pyrimidine synthesis leading to a build up of orotate. Following the synthesis of carbamoyl phosphate, a series of subsequent reactions yield uracil monosphosphate, which is the intermediate of pyrimidine synthesis.

UMP, much like IMP, serves as the intermediate to pyrimidine synthesis and can undergo sequential phosphorylation to form UTP, which can be converted to cytidine (CTP). Alternatively, UMP can be converted to a deoxy form (dUDP) to be used as substrate for the synthesis of thymidine. The conversion of dUDP to dTMP is catalyzed by thymidylate synthase, which requires folate (N5,N10 methylene tetrahydrofolate) as a methyl and hydrogen donor to complete this conversion (figure 7.13).

dUMP arrow enzyme thymidylate synthase to dTMP arrow with ATP arrow ADP and enzyme kinase to dTDP arrow with ATP arrow ADP and enzyme kinase to dTTP. Circular diagram attached to arrow between dUMP, to dihydrofolate arrow with NADPH arrow NADP+ and enzyme DHFR to THF arrow with Serine arrow glycine and enzyme SHMT to N5, N10 methylene-THF.
Figure 7.13: Interaction of thymidylate synthesis with the folate cycle. SHMT: serine hydroxymethyltransferase; DHFR: dihydrofolate reductase.

Defects in pyrimidine synthesis most commonly present as an increase in orotic acid in the urine. Deficiencies in the attachment of PRPP to orotate (or the decarboxylation of orotate monosphosphate) can result in the accumulation of orotic acid; similarly deficiencies of the urea cycle, which lead to an accumulation of carbamoyl phosphate, can increase flux through pyrimidine synthesis and cause an increase in orotic acid. Accumulation of orotic acid is used as a clinical indicator of pyrimidine deficiencies or deficiencies in the urea cycle.

Regulation of pyrimidine synthesis

The reaction catalyzed by CSPII is the regulatory step in the pathway and is activated by PRPP and ATP and inhibited by UTP.

Clinical importance of folate cycle inhibitors and synthesis of dTMP

Synthesis of dTMP for DNA synthesis is the rate-limiting step for the replication process, and therefore disruption of this conversion is very effective at reducing cellular proliferation. Inhibition of thymidylate synthase by 5-fluorouracil (5-FU) is a common anticancer treatment. 5-FU functions as a thymine analog and will irreversibly bind the enzyme. Similarly, methotrexate is an inhibitor of dihyrofolate reductase (DHFR), which is part of the folate cycle needed to reduce dihydrofolate to tetrahydrofolate. Inhibition of this process reduces substrate needed for the thymidylate synthase reaction and has a similar effect as inhibition of by 5-FU (figure 7.13).

Summary of pathway regulation

Metabolic pathway Major regulatory enzyme Allosteric effectors Hormonal effects
Pyrimidine synthesis CPSII PRPP, ATP (+), UTP (-) None
Purine synthesis PRPP synthetase Pi (+), Purine bases (-) Note: PRPP synthetase is required for both purine and pyrimidine synthesis
Purine synthesis GPAT PRPP (+), IMP, AMP, GMP (-) Note: PRPP synthetase is required for both purine and pyrimidine synthesis

Table 7.2: Summary of pathway regulation.

7.2 References and resources

Text

Ferrier, D. R., ed. Lippincott Illustrated Reviews: Biochemistry, 7th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, 2017, Chapter 13: Pentose Phosphate Pathway and NAPDH, Chapter 22: Nucleotide Metabolism.

Le, T., and V. Bhushan. First Aid for the USMLE Step 1, 29th ed. New York: McGraw Hill Education, 2018, 35–37, 79.

Lieberman, M., and A. Peet, eds. Marks’ Basic Medical Biochemistry: A Clinical Approach, 5th ed. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins, 2018, Chapter 27: Pentose Phosphate Pathway, Chapter 39: Purine and Pyrimidine Synthesis.

Figures

Grey, Kindred, Figure 7.5 Overview of purine and pyrimidine bases. 2021. Chemical structure by Henry Jakubowski. https://archive.org/details/7.5_20210926. CC BY 4.0.

Grey, Kindred, Figure 7.6 Synthesis of PRPP and regulation of PRPP synthetase. 2021. https://archive.org/details/7.6_20210926. CC BY 4.0.

Grey, Kindred, Figure 7.7 Overview of purine synthesis. The reaction catalyzed by GPAT is the regulatory enzyme of the pathway. 2021. https://archive.org/details/7.7_20210926. CC BY 4.0.

Grey, Kindred, Figure 7.8 Purine synthesis and regulation of glutamine:phosphoribosylpyrophosphate amidotransferase. 2021. https://archive.org/details/7.8_20210926. CC BY 4.0.

Grey, Kindred, Figure 7.9 Breakdown of nucleotides. 2021. https://archive.org/details/7.9_20210926. CC BY 4.0.

Grey, Kindred, Figure 7.10 Nucleotide base salvage. Reaction catalyzed by HGPRT is clinically relevant as deficiencies can cause accumulation of uric acid. 2021. https://archive.org/details/7.10_20210926. CC BY 4.0.

Grey, Kindred, Figure 7.11 Nucleotide specific pathways for base salvage. 2021. https://archive.org/details/7.11_20210926. CC BY 4.0.

Grey, Kindred, Figure 7.12 Overview of pyrimidine synthesis. The reaction catalyzed by carbamoyl phosphate synthetase I is the regulatory enzyme of the pathway. 2021. https://archive.org/details/7.12_20210926. CC BY 4.0.

Grey, Kindred, Figure 7.13 Interaction of thymidylate synthesis with the folate cycle. SHMT: Serine hydroxymethyltransferase; DHFR: Dihydrofolate reductase. 2021. https://archive.org/details/7.13_20210926. CC BY 4.0.

Lieberman M, Peet A. Figure 7.4 Basic structure of nucleotides. Adapted under Fair Use from Marks’ Basic Medical Biochemistry. 5th Ed. pp 216. Figure 12.3 Nucleoside and nucleotide structures displayed with ribose as the sugar. 2017. Chemical structure by Henry Jakubowski.