Medical Biochemistry

CHAPTER 31

Biosynthesis and Degradation of Nucleotides

Alejandro Gugliucci and Robert Thornburg

Learning objectives

After reading this chapter you should be able to:

image Compare and contrast the structure and biosynthesis of purines and pyrimidines, highlighting differences between de novo and salvage pathways.

image Describe how cells meet their requirements for nucleotides at various stages in their cell cycle.

image Explain the biochemical rationale for using fluorouracil and methotrexate in chemotherapy.

image Describe the metabolic basis and therapy for classic disorders in nucleotide metabolism: gout, Lesch–Nyhan syndrome and SCIDS.

Introduction

Nucleotides, molecules composed of a pentose, a nitrogenous base and phosphate, are key elements in cell physiology since they are:

image precursors of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA);

image components of coenzymes, e.g NAD(H), NADP(H), FMN(H2), and CoA;

image energy currency, driving many metabolic processes, e.g. ATP and GTP;

image carriers in biosynthesis, e.g. UDP for carbohydrates and CDP for lipids;

image modulators of allosteric regulation of metabolism;

image second messengers, e.g. cAMP and cGMP.

We can synthesize ample amounts of purine and pyrimidine nucleotides from metabolic intermediates. In this way, although we ingest dietary nucleic acids and nucleotides, survival does not require their absorption and utilization. Because nucleotides are involved in so many levels of metabolism they are important targets for chemotherapeutic agents used in treatment of microbial and parasitic infections and cancer.

This chapter will describe the structure and metabolism of the two classes of nucleotides: purines and pyrimidines. The metabolic pathways are divided into four sections:

image De novo synthesis of nucleotides from basic metabolites, which is required in growing cells.

image Salvage pathways that recycle preformed bases and nucleosides and provide an adequate supply of nucleotides for cells at rest.

image Catabolic pathways for excretion of nucleotide degradation products, a process that is essential to limit the accumulation of toxic levels of nucleotides within cells: impaired elimination or increased production of uric acid may produce gout.

image Biosynthetic pathways for conversion of the ribonucleotides into the deoxyribonucleotides, providing precursors for DNA.

Purines and pyrimidines

Nucleotides are formed from three components: a nitrogenous base, a five-carbon sugar, and phosphate

The nitrogenous bases found in nucleic acids belong to one of two heterocyclic groups, either purines or pyrimidines (Fig. 31.1). The major purines of both DNA and RNA are guanine and adenine. In DNA, the major pyrimidines are thymine and cytosine, while in RNA, they are uracil and cytosine; thymine is unique to DNA and uracil is unique to RNA.

image

FIG. 31.1 Classification of nucleotides.
Basic structure of purines and pyrimidines.

When the nitrogenous bases are combined with a five-carbon sugar, they are known as nucleosides. When the nucleosides are phosphorylated, the compounds are known as nucleotides. The phosphate can be attached either at the 5′position or the 3′-position of ribose, or both. Table 31.1 gives the names and structures of the most important purines and pyrimidines.

Table 31.1

Names and structures of important purines and pyrimidines

image

The designation NTP refers to the ribonucleotide. The prefix d, as in dATP, is used to identify deoxyribonucleotides. dTTP is usually written as TTP, with the d-prefix implied.

Purine metabolism

De novo synthesis of the purine ring: synthesis of inosine monophosphate (IMP)

Purines and pyrimidines are synthesized by both de novo and salvage pathways

The demand for nucleotide biosynthesis can vary greatly. It is high during the S-phase of the cell cycle, when cells are about to divide (Chapter 42). The process is therefore very active in growing tissues, actively proliferating cells like blood cells and cancer cells, and when tissues are regenerating. Purine and pyrimidine biosynthesis are energetically expensive processes that are subject to intracellular mechanisms that sense and effectively regulate the pool sizes of intermediates and products.

The raw materials for purine synthesis are: CO2, nonessential amino acids (Asp, Glu, Gly), and folic acid derivatives which act as single carbon donors. Five molecules of ATP are needed for the synthesis of IMP, the first purine product and common precursor of AMP and GMP. The starting material for synthesis of IMP is ribose 5-phosphate, a product of the pentose phosphate pathway (Chapter 12). The first step, catalyzed by ribose phosphate pyrophosphokinase (PRPP synthetase), generates the activated form of the pentose phosphate by transferring a pyrophosphate group from ATP to form 5-phosphoribosyl-pyrophosphate (PRPP) (Fig. 31.2). In a series of 10 reactions, PRPP is converted to IMP. Most of the carbons and all of the nitrogens of the purine ring are derived from the amino acids; one carbon is derived from CO2 and two from N10-formyltetrahydrofolate (THF), a derivative of folic acid. Folate deficiency can impair purine synthesis, which can produce disease or can be exploited clinically to kill rapidly dividing cells, which have a high demand for purine biosynthesis. The end product of this sequence of reactions is the ribonucleotide IMP; the nucleoside is inosine and the purine base is called hypoxanthine.

image

FIG. 31.2 Synthesis of IMP.
*The asterisk identifies the regulatory enzyme amidophosphoribosyl transferase (2).

Synthesis of ATP and GTP from IMP

IMP does not accumulate significantly within the cell. As shown in Figure 31.3, it is converted to both AMP and GMP. Two enzymatic reactions are required in each case (see Fig. 31.3). Distinct enzymes, adenylate kinase and guanylate kinase, use ATP to synthesize the nucleotide diphosphates from the nucleotide monophosphates. Finally, a single enzyme, termednucleotide diphosphokinase, converts diphosphonucleotides into nucleotide triphosphates. This enzyme has activity towards all nucleotide diphosphates, including pyrimidines and purines and both ribo- and deoxyribonucleotides for synthesis of RNA and DNA, respectively.

image

FIG. 31.3 Conversion of IMP into AMP and GMP.
Two enzymatic reactions are needed in each branch of the pathway. XMP, xanthosine monophosphate.

Salvage pathways for purine nucleotide biosynthesis

In addition to de novo synthesis, cells can use preformed nucleotides obtained from the diet or from the breakdown of endogenous nucleic acids through salvage pathways. In mammals, there are two enzymes in the purine salvage pathway. Adenine phosphoribosyl transferase (APRT) converts free adenine into AMP (Fig. 31.4A). Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) catalyzes a similar reaction for both hypoxanthine (the purine base in IMP) and guanine (Fig. 31.4B). Purine nucleotides are synthesized preferentially by salvage pathways, so long as the free nucleobases are available. This preference is mediated by hypoxanthine inhibition of amidophosphoribosyl transferase, step 2 of the de novo pathway. Note that step 2 is the site of inhibition of purine biosynthesis, since PRPP is also used in other biosynthetic processes, including nucleotide salvage pathways.

image

FIG. 31.4 The purine salvage pathways.
(A) adenine phosphoribosyl transferase. (B) Hypoxanthine-guanine phosphoribosyl transferase.

Purine and uric acid metabolism in humans

Sources and disposal of uric acid

Uric acid is the endproduct of purine catabolism in humans

Uric acid, the end product of purine catabolism in humans, is not metabolized and must be excreted. However, the complex renal handling of urate, described below, suggests an evolutionary advantage to having high circulating levels of urate. As noted in Chapter 37, uric acid is a circulating antioxidant. At pH 7.4, it is 98% ionized and therefore circulates as monosodium urate. This salt has poor solubility; the extracellular fluid becoming saturated at urate concentrations little above the upper limit of the reference range. Therefore, there is a tendency for monosodium urate to crystallize in subjects with hyperuricemia. The most obvious clinical manifestation of this process is gout, in which crystals form in cartilage, synovium and synovial fluid. This can be accompanied by renal calculi (urate stones) and tophi (accumulation of sodium urate in soft tissues). A sudden increase in urate production, for example during chemotherapy when many cells die rapidly, can lead to widespread crystallization of urate in joints, but mainly in urine, causing an acute urate nephropathy.

There are three sources of purines in man: de novo synthesis, salvage pathways, and diet. The body urate pool (and thus plasma uric acid concentration) is governed by the relative rates of urate formation and excretion. Over half of urate is excreted by the kidney, the rest by the intestines, where bacteria dispose of it. In the kidney, urate is filtered and almost totally reabsorbed in the proximal tubule. Distally, both secretion and absorption occur, so that overall urate clearance amounts to about 10% of the filtered load, i.e. 90% is retained in the body. Normally, urate excretion increases if the filtered load is increased. Because of the role of the kidney in urate metabolism, kidney diseases can lead to urate retention and urate precipitation in the kidney (stones) and urine. Dietary purines account for about 20% of excreted urate. Therefore, restricting purines in the diet (less meat and red wine) can reduce urate levels by only 10–20%.

image Advanced concept box Salvage pathways are the principal source of nucleotides in lymphocytes

In humans, resting T lymphocytes, immune system cells produced in the thymus (Chapter 38), meet their routine metabolic requirements for nucleotides through the salvage pathway, but de novo synthesis is required to support growth of rapidly dividing cells. The salvage of nucleotides is especially important in HIV-infected T lymphocytes. In asymptomatic patients, resting lymphocytes show a block in de novo pyrimidine biosynthesis, and correspondingly reduced pyrimidine pool sizes. Following activation of the T-lymphocyte population, these cells cannot synthesize sufficient new DNA. The activation process leads to cell death, contributing to the decline in the T-lymphocyte population during the late stages of HIV infection.

The salvage pathways are especially important for many parasites as well. These organisms prey metabolically on their host, using preformed metabolites, including nucleotides. Some parasites, such as MycoplasmaBorrelia, and Chlamydia, have lost the genes required for the de novo synthesis of nucleotides; they obtain these important components from their host.

image Clinical box Gout results from the excess of uric acid

Diagnosis.

The diagnosis of gout is primarily clinical and supported by the demonstration of hyperuricemia. About 90% of patients appear to excrete urate at an inappropriately low rate for the plasma concentration, while about 10% have excessive production. Gouty arthritis has a typically hyperacute onset (less than 24 h), with severe pain, swelling, redness and warmth in the joint(s), characteristically in the big toe. It is confirmed by the presence of tophi or sodium urate crystals in the synovial fluid. The crystals are needle-shaped, are seen inside neutrophils and have negative birefringence when viewed with polarized light.

Pathogenesis.

Urate crystals in joints are phagocytized by neutrophils (leukocytes in blood and tissues). The crystals damage lysosomal and cellular membranes, causing cellular disruption and death. Release of lysosomal enzymes in the joint precipitates an acute inflammatory reaction. Several cytokines enhance and perpetuate the inflammation and phagocytic cells, monocytes and macrophages aggravate the inflammation.

Management.

The acute attack is managed with antiinflammatory agents, including NSAIDs. Dietary changes (less meat, increased water intake, weight reduction) and changes in concurrent drug therapies, such as diuretics, may be helpful. Probenecid, a uricosuric drug, is commonly employed to reduce uricemia. Colchicine, a microtubule disruptor, may also be used during an acute attack to inhibit phagocytosis and inflammation. If the patient is already a hyperexcretor, or tophi or renal disease are present, then allopurinol is used. Allopurinol is an inhibitor of xanthine oxidase (Fig. 31.5). Allopurinol undergoes the first oxidation to yield alloxanthine, but cannot undergo the second oxidation. Alloxanthine remains bound to the enzyme, acting as a potent competitive inhibitor. This leads to reduced formation of uric acid and accumulation of xanthine and hypoxanthine, which are more soluble and are excreted in urine.

image

FIG. 31.5 Degradation of purines and biochemical basis of allopurinol treatment of gout.
Inhibition of xanthine oxidase (XO) by alloxanthine is the mechanism involved in allopurinol treatment of gout. The enzyme uricase is missing in primates (including humans) but is commonly used for measurement of serum uric acid levels in humans. (1) 5′-nucleotidase; (2) adenosine deaminase; (3) AMP deaminase; (4) purine nucleotide pyrophosphorylase; (5) guanine deaminase.

Endogenous formation of uric acid

Each of the purine monophosphates (IMP, GMP and AMP) can be converted into their corresponding nucleosides by 5′-nucleotidase. The enzyme purine nucleoside phosphorylase then converts the nucleosides inosine or guanosine into the free purine bases hypoxanthine and guanine, and ribose-1-P. Hypoxanthine is oxidized and guanine is deaminated to yield xanthine (Fig. 31.5). Two other enzymes, AMP deaminase and adenosine deaminase, convert the amino group of AMP and adenosine into IMP and inosine, respectively, which are then converted to hypoxanthine. In effect, guanine is directly converted to xanthine, while inosine and adenine are converted to hypoxanthine, then to xanthine.

Xanthine oxidase (XO), the final enzyme in this pathway, catalyzes a two-step oxidation reaction, converting hypoxanthine to xanthine, then xanthine to uric acid. Uric acid is the final metabolic product of purine catabolism in primates, birds, reptiles, and many insects. Other organisms, including most mammals, fish, amphibians and invertebrates, metabolize uric acid to more soluble products, such as allantoin (see Fig. 31.5).

Hyperuricemia and gout

Most persons with hyperuricemia remain asymptomatic throughout life, but there is no gout without hyperuricemia

Plasma urate concentration is, on average, higher in men than in women, tends to rise with age, and is usually elevated in obese subjects and subjects in the higher socioeconomic groups. Higher levels of uric acid correlate with high sugar consumption. The risk of gout, a painful disease resulting from precipitation of sodium urate crystals in joints and dermis, increases with higher plasma urate concentrations (see Box on p. 414). Hyperuricemia can occur due to increased formation or decreased excretion of uric acid, or both. Decreased renal excretion of urate can result from a decrease in filtration and/or secretion. Many factors (including drugs and alcohol) also affect tubular handling of urates and can cause or increase hyperuricemia.

Pyrimidine metabolism

As with the purines, the pyrimidines (uracil, cytosine and thymine) are also synthesized through a complex series of reactions using raw materials readily available in cells. One important difference is that the pyrimidine base is made first and the sugar added late (Fig. 31.6), whereas purines are assembled on a ribose-5-P scaffold (Fig. 31.2). Uridine monophosphate (UMP) is the precursor of all pyrimidine nucleotides. The de novo pathway produces UMP, which is then converted to cytidine triphosphate (CTP) and thymidine triphosphate (TTP). Salvage pathways also recover preformed pyrimidines.

image

FIG. 31.6 The metabolic pathway for synthesis of pyrimidines.
Formation of orotic acid and UMP, the first pyrimidine nucleotide.

image Clinical box Lesch–nyhan syndrome – HGPRT deficiency

The gene encoding HGPRT is located on the X-chromosome. Its deficiency results in a rare, X-linked recessive disorder, Lesch–Nyhan syndrome. The lack of HGPRT causes an overaccumulation of PRPP, which is also the substrate for the enzyme amidophosphoribosyl transferase. This stimulates purine biosynthesis by up to 200-fold. Because of increased purine synthesis, the degradation product, uric acid, also accumulates to high levels. Elevated uric acid leads to a crippling gouty arthritis and severe neuropathology, resulting in mental retardation, spasticity, aggressive behavior, and a compulsion towards self-mutilation by biting and scratching.

De novo pathway

Pyrimidine and purine nucleoside biosynthesis share several common precursors: CO2, amino acids (Asp, Gln), and, for thymine, N5,N10-methylene-THF. The pathway for biosynthesis of UMP is outlined in Figure 31.6. The first step, catalyzed by the enzyme carbamoyl phosphate synthetase II (CPS II), uses bicarbonate, glutamine, and 2 moles of ATP to form carbamoyl phosphate (CPS I is used in the synthesis of arginine in the urea cycle; Chapter 19). Most of the atoms required for formation of the pyrimidine ring are derived from aspartate, added in a single step by aspartate transcarbamoylase (ATCase). Carbamoyl aspartate is then cyclized to dihydroorotic acid by the action of the enzyme dihydroorotase. Dihydroorotic acid is oxidized to orotic acid by a mitochondrial enzyme, dihydroorotate dehydrogenase. Leflunomide, a specific inhibitor of this enzyme, is used for treatment of rheumatoid arthritis because blockage of this step inhibits lymphocyte activation and thereby limits inflammation. The ribosyl-5′-phosphate group from PRPP is then transferred onto orotic acid to form orotidine monophosphate (OMP). Finally, OMP is decarboxylated to form UMP. UTP is synthesized in two enzymatic phosphorylation steps by the actions of UMP kinase and nucleotide diphosphokinase. CTP synthetase converts UTP into CTP by amination of UTP (Fig. 31.7). This completes the synthesis of the ribonucleotides for synthesis of RNA.

image

FIG. 31.7 Synthesis of pyrimidine triphosphates.
Synthesis of thymidine is inhibited by fluorodeoxyuridylate (FdUMP), methotrexate, aminopterin and trimethoprim at the indicated sites.

Metabolic channeling by multienzymes improves efficiency

In bacteria, the six enzymes of pyrimidine (UMP) biosynthesis exist as distinct proteins. However, during the evolution of mammals the first three enzymatic activities have been fused together into CAD, a single multifunctional polypeptide encoded by a single gene. The name of the enzyme derives from its three activities: Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, and Dihydroorotase. The final two enzymatic activities of pyrimidine biosynthesis, orotate phosphoribosyl transferase and orotidylate decarboxylase, are also fused into a single enzyme, UMP synthase. As with the fatty acid synthase complex (Chaper 16), this fusion of sequential enzyme activities avoids the diffusion of the metabolic intermediates into the intracellular milieu, thereby improving the metabolic efficiency of the individual steps.

Pyrimidine salvage pathways

As with the purines, free pyrimidine bases, available from the diet or from the breakdown of nucleic acids, can be recovered by several salvage enzymes. Uracil phosphoribosyl transferase (UPRTase) is similar to the enzymes of the purine salvage pathways. This also activates some chemotherapeutic agents such as 5-fluorouracil (FU) or 5-fluorocytosine (FC). A uridine-cytidine kinase and a more specific thymidine kinase catalyze the phosphorylation of these nucleosides; nucleotide kinases and diphosphokinase complete the salvage process.

Formation of deoxynucleotides

Ribonucleotide reductase

Ribonucleotide reductase catalyzes reduction of ribose to deoxyribose in nucleotides for synthesis of DNA

Because DNA uses deoxyribonucleotides instead of the ribonucleotides found in RNA, cells require pathways to convert ribonucleotides into the deoxy forms. The adenine, guanine and uracil deoxyribonucleotides are synthesized from their corresponding ribonucleotide diphosphates by direct reduction of the 2′-hydroxyl by the enzyme ribonucleotide reductase, as shown for dUDP in Figure 31.7. The reduction of the 2′-hydroxyl of ribose uses a pair of protein-bound sulfhydryl groups (cysteine residues). The hydroxyl group is released as water, and the cysteines are oxidized to cystine during the reaction. To regenerate an active enzyme, the disulfide must be reduced back to the original sulfhydryl pair by disulfide exchange; this is accomplished by reaction with a small protein, thioredoxin. The thioredoxin, a highly conserved Fe-S protein, is in turn reduced by the flavoprotein, thioredoxin reductase.

A unique pathway to TTP

Thymine is synthesized by a complex reaction pathway, providing many opportunities for chemotherapy

The nucleotide deoxy-TMP, abbreviated as TMP because thymine is unique to DNA, is synthesized by a special pathway involving methylation of the deoxyribose form of uridylate, dUMP (Fig. 31.7). The TMP biosynthetic pathway leads from UMP to UDP, then, through ribonucleotide reductase, to dUDP. The dUDP is then phosphorylated to dUTP, which creates an unexpected biochemical problem. DNA polymerase does not effectively discriminate between the two deoxyribonucleotides, dUTP and TTP – the only difference is a methyl group atC-5. It incorporates dUTP into DNA in vitro; this reaction would lead to high rates of mutagenesis in vivo. Therefore, cells limit the concentration of dUTP by rapidly hydrolyzing dUTP to dUMP with the enzyme dUTPase. This enzyme cleaves a high-energy bond and releases pyrophosphate, which is rapidly hydrolyzed to phosphate, shifting the equilibrium ever further towards the formation of dUMP. The dUMP is converted to TMP by thymidylate synthase (TS), using N5,N10-methylene-THF as the methyl donor; the dihydrofolate product is recycled by action of the enzymes dihydrofolate reductase and serine hydroxymethyl transferase. Two rounds of phosphorylation of TMP yield TTP for synthesis of DNA.

The synthesis of TTP is a roundabout pathway, but provides opportunities for chemotherapy through inhibition of TMP biosynthesis (see Fig. 31.7). There is only one reaction in pyrimidine synthesis that requires a THF derivative: conversion of dUMP to TMP, catalyzed by thymidylate synthase. This reaction is often rate limiting for cell division. Indeed, folate deficiency impairs cell replication, especially the replication of rapidly dividing cells. Thus, folate deficiency is a frequent cause of anemia: bone marrow cells involved in erythropoiesis and hematopoiesis are among the most rapidly dividing cells in the body. As outlined in the Advanced Concept Box on the next page, inhibition of thymidylate synthase, either directly or by inhibition of THF recycling, provides a special opportunity for chemotherapy,

image

FIG. 31.8 Formation of deoxyribonucleotides, except TTP, by ribonucleotide reductase.
Thioredoxin and NADPH (from the pentose phosphate pathway) are required for recycling of the enzyme.

targeting synthesis of DNA precursors in rapidly dividing cancer cells.

image Advanced concept box Chemotherapeutic targets: folate recycling and thymidylate synthase

Fluorodeoxyuridylate (FdUMP) is a specific, suicide inhibitor of thymidylate synthase. In FdUMP, a highly electronegative fluorine replaces the carbon-5 proton of uridine. This compound can begin the enzymatic conversion into dTMP by forming the enzyme–FdUMP covalent complex; however, the covalent intermediate cannot accept the donated methyl group from methylene THF, nor can it be broken down to release the active enzyme. The result is a suicide complex in which the substrate is covalently locked at the active site of thymidylate synthase. The drug is frequently administered as flurouracil, and the body's normal metabolism converts the fluorouridine into FdUMP. Fluorouracil is used against breast, colorectal, gastric, and uterine cancers.

Fluorocytosine is a potent antimicrobial agent. Its mechanism of action is similar to that of FdUMP; however, it must first be converted into fluorouracil by the action ofcytosine deaminase. The fluorouracil is subsequently converted into FdUMP, which blocks thymidylate synthase as above. While cytosine deaminase is present in most fungi and bacteria, it is absent in animals and plants. Therefore, in humans, fluorocytosine is not converted into fluorouracil and is nontoxic, while in the microbes, metabolism of fluorocytosine results in cell death.

Aminopterin and methotrexate are folic acid analogues that bind about 1000-fold more tightly to dihydrofolate reductase (DHFR) than does dihydrofolate. In this manner, they competitively, almost irreversibly, block the synthesis of dTMP. These compounds are also competitive inhibitors of other THF-dependent enzyme reactions used in the biosynthesis of purines, histidine, and methionine. Trimethoprim binds to DHFR, and binds more tightly to bacterial DHFRs than it does to mammalian enzymes, making it an effective antibacterial agent. Folate analogues are relatively nonspecific chemotherapeutic agents. They poison rapidly dividing cells, not just cancer cells but also hair follicles and gut epithelial cells, causing the loss of hair and the gastrointestinal side effects of chemotherapy.

De novo nucleotide metabolism is highly regulated

Ribonucleotide reductase is the allosteric enzyme that coordinates a balanced supply of deoxynucleotides for synthesis of DNA

Because nucleotides are required for mammalian cells to proliferate, the enzymes involved in de novo synthesis of both purines and pyrimidines are induced during the S-phase of cell division. Covalent and allosteric regulation also plays an important role in control of nucleotide synthesis. The multimeric protein CAD is activated by phosphorylation by protein kinases in response to growth factors, increasing its affinity for PRPP and decreasing inhibition by UTP. Both of these changes favor biosynthesis of pyrimidines for cell division.

Mole per mole, pyrimidine biosynthesis parallels purine biosynthesis, suggesting the presence of a coordinated control. Among them, one of the key points is the PRPP synthase reaction. PRPP is a precursor for all the ribo- and deoxyribonucleotides. PRPP synthase is inhibited by both pyrimidine and purine nucleotides.

Ribonucleotide reductase coordinates the biosynthesis of all four deoxynucleotides

Because a single enzyme is responsible for the conversion of all ribonucleotides into deoxyribonucleotides, this enzyme is subject to a complex network of feedback regulation. Ribonucleotide reductase contains several allosteric sites for metabolic regulation. Levels of each of the dNTPs modulate the activity of the enzyme toward the other NDPs. By regulating the enzymatic activity of deoxyribonucleotide synthesis as a function of the concentration of the different dNTPs, often described as ‘cross-talk’ between the pathways, the cell insures that the proper ratios of the different deoxyribonucleotides are produced for normal growth and cell division.

Catabolism of pyrimidine nucleotides

In contrast to the degradation of purines to uric acid, pyrimidines are degraded to readily soluble compounds, which are readily eliminated in urine and are not a frequent source of pathology. Orotic acidurias may occur in the rare cases when enzymes in the pyrimidine catabolic pathways are defective. The pyrimidine nucleotides and nucleosides are converted to the free bases and the heterocyclic ring is cleaved, yielding β-aminoisobutyrate as the main excretion product, plus some ammonia and CO2.

Summary

image Nucleotides are synthesized primarily from amino acid precursors and phosphoribosyl pyrophosphate by complex, metabolically expensive, multistep pathways.

image De novo nucleotide metabolism is required for cell proliferation, but salvage pathways also play a prominent role in nucleotide metabolism.

image Both classes of nucleotides (purines and pyrimidines) are synthesized as precursors (IMP, UMP), which are then converted into the DNA precursors (dATP, dGTP, dCTP, TTP).

image With the exception of TTP, ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductase. TTP is synthesized from dUMP by a special pathway involving folates.

image Salvage pathways have proven useful for the activation of pharmaceutical agents, while the uniqueness of the pathway for synthesis of TTP has provided a special target for chemotherapeutic inhibition of DNA synthesis and cell division in cancer cells.

image High plasma concentrations of uric acid, the final product of purine catabolism in man, can lead to gout and kidney stones.

image Clinical box Severe combined immunodeficiency syndromes (SCIDS) are caused by impaired purine salvage pathways

SCIDS are a group of fatal disorders resulting from defects in both cellular and humoral immune function. SCIDS patients cannot efficiently produce antibodies in response to an antigenic challenge. Approximately 50% of patients with the autosomal recessive form of SCIDS have a genetic deficiency in the purine salvage enzyme adenosine deaminase. The pathophysiology involves lymphocytes of both thymic and bone marrow origin (T and B lymphocytes), as well as ‘self-destruction’ of differentiated cells following antigen stimulation. The precise cause of cell death is not yet known, but may involve accumulation in lymphoid tissues of adenosine, deoxyadenosine and dATP, accompanied by ATP depletion. dATP inhibits ribonucleotide reductase and therefore impedes DNA nucleotide synthesis. The finding that deficiency of the next enzyme in the purine salvage pathway, nucleoside phosphorylase, is also associated with an immune deficiency disorder suggests that integrity of the purine salvage pathway is critical for normal differentiation and function of immunocompetent cells in man.

image Advanced concept box The yin and yang of xanthine oxidase – beyond uric acid production

Xanthine oxidase (XO) is a ubiquitous cytosolic flavoprotein which controls the rate-limiting step in purine catabolism. The oxidation of xanthine to uric acid produces FADH2, and the reoxidation of FADH2 produces reactive oxygen species (ROS; superoxide and hydrogen peroxide), which are toxic at high concentrations in the cell. An excess of ROS production is associated with many acute and chronic diseases, including ischemia-reperfusion injury (IRI), cardiovascular disease, microvascular syndromes, metabolic syndrome and cancer (Chapter 37). XO, among other enzymes, contributes to the generation of excess ROS in these conditions. In IRI, the ROS are produced during reperfusion of the tissue, i.e. during the recovery phase. Allopurinol, an XO inhibitor, is being evaluated as adjuvant therapy to limit ROS production during recovery from myocardial infarction and stroke. In contrast, XO is also being conjugated to antitumor antibodies in experimental studies to direct ROS production to the tumor environment in order to kill cancer cells. Thus, while XO is a lead actor in purine catabolism, it also plays other roles in pathology and therapy.

image Clinical box Reverse evolution: uricase as a new treatment for refractory gout

Most of the drugs employed to treat patients with gout have been used for over 40 years. More recently, as our physiologic understanding of gout has improved, new innovative treatments have been developed, such as enzyme therapy by administration of uricase. Pegloticase, a recombinant uricase, is a novel treatment option for patients suffering from chronic and refractory gout. Human trials show that pegloticase maintains uric acid concentrations below 7 mg/dL in patients with chronic gout. These recombinant uricases may have a place in the treatment of gout, particularly in patients with severe and tophaceous gout to promote tophi dissolution. Pegloticase is an effective option for patients with symptomatic gout for whom existing hypouricemic agents are unsuccessful or contraindicated.

Active learning

1. Compare the roles of de novo synthesis and salvage pathways of nucleotide synthesis in various cell types, e.g. erythrocytes, lymphocytes, muscle and liver.

2. In addition to its activity as a xanthine oxidase inhibitor, what other activities of allopurinol might contribute to its efficacy for treatment of gout?

3. Discuss the use of thymidylate synthetase inhibitors and folate analogues for treatment of diseases other than cancer, e.g. arthritis, psoriasis.

Further reading

Agarwal, A, Banerjee, A, Banerjee, UC. Xanthine oxidoreductase: a journey from purine metabolism to cardiovascular excitation-contraction coupling. Crit Rev Biotechnol. 2011; 31:264–280.

Fang, J, Nakamura, H, Iyer, AK. Tumor-targeted induction of oxystress for cancer therapy. J Drug Target. 2007; 15:475–486.

Gangjee, A, Jain, HD, Kurup, S. Recent advances in classical and non-classical antifolates as antitumor and antiopportunistic infection agents. Anticancer Agents Med Chem. 2008; 8:205–231.

Garay, RP, El-Gewely, MR, Labaune, JP, Richette, P. Therapeutic perspectives on uricases for gout. Joint Bone Spine. 2012; 79:237–242.

Jordan, KM. Up-to-date management of gout. Curr Opin Rheumatol. 2012; 24:145–151.

Jurecka, A. Inborn errors of purine and pyrimidine metabolism. J Inherit Metab Dis. 2009; 32:247–263.

Lee, BE, Toledo, AH, Anaya-Prado, R, et al. Allopurinol, xanthine oxidase, and cardiac ischemia. J Investig Med. 2009; 57:902–909.

Nyhan, WL. The recognition of Lesch–Nyhan syndrome as an inborn error of purine metabolism. J Inherited Metab Dis. 1997; 20:171–178.

Shannon, JA, Cole, SW. Pegloticase: a novel agent for treatment-refractory gout. Ann Pharmacother. 2012; 46:368–376.

Sigoillot, FD, Berkowski, JA, Sigoillot, SM, et al. Cell cycle-dependent regulation of pyrimidine biosynthesis. J Biol Chem. 2003; 278:3403–3409.

Websites

Pictures of Gout. http://www.mediscan.co.uk/cfm/resultssearch.cfm?action=keyword&log=nk.

Gout. www.niams.nih.gov/Health_Info/Gout/default.asp.

Lesch–Nyhan. http://emedicine.medscape.com/article/1181356-overview.

Purine and pyrimidine metabolism disorders. http://www.merckmanuals.com/professional/pediatrics/inherited_disorders_of_metabolism/purine_and_pyrimidine_metabolism_disorders.html#v1103920.

SCIDS. www.scid.nethttp://themedicalbiochemistrypage.org/nucleotide-metabolism.php.