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Introduction

 

 

 

 

 

 

 

 

 

 

 

The metabolic requirements for the nucleotides and their cognate bases can be met by both dietary intake or synthesis de novo from low molecular weight precursors. Indeed, the ability to salvage nucleotides from sources within the body alleviates any nutritional requirement for nucleotides, thus the purine and pyrimidine bases are not required in the diet. The salvage pathways are a major source of nucleotides for synthesis of DNA, RNA and enzyme co-factors.

Extracellular hydrolysis of ingested nucleic acids occurs through the concerted actions of endonucleases, phosphodiesterases and nucleoside phosphorylases. Endonucleases degrade DNA and RNA at internal sites leading to the production of oligonucleotides. Oligonucleotides are further digested by phosphodiesterases that act from the ends inward yielding free nucleosides. The bases are hydrolyzed from nucleosides by the action of phosphorylases that yield ribose-1-phosphate and free bases. If the nucleosides and/or bases are not re-utilized the purine bases are further degraded to uric acid and the pyrimidines to β-aminoiosobutyrate, NH3 and CO2.

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Activation of Ribose-5-Phosphate

Both the salvage and de novo synthesis pathways of purine and pyrimidine biosynthesis lead to production of nucleoside-5'-phosphates through the utilization of an activated sugar intermediate and a class of enzymes called phosphoribosyltransferases. The activated sugar used is 5-phosphoribosyl-1-pyrophosphate, PRPP. PRPP is generated by the action of PRPP synthetase (also called ribose-phosphate pyrophosphokinase 1) and requires energy in the form of ATP. At least three different enzymes with PRPP synthetase activity have been identified which are encoded by three distinct genes. These genes are identified as PRPS1, PRPS2, and PRPS1L1 (PRPS1-like 1). The PRPS1 and PRPS2 genes are both located on the X chromosome, PRPS1 is on the q arm (Xq22.3) and PRPS2 is on the p arm (Xp22.2). The PRPS1 gene is composed of 7 exons that generate two alternatively spliced mRNAs encoding isoform 1 (318 amino acids) and isoform 2 (114 amino acids). Mutations in the PRPS1 gene are those that are associated with PRPP synthetase superactivity. In addition, mutations in the PRPS1 gene are associated with Arts syndrome and Charcot-Marie-Tooth disease X-linked recessive type 5 (CMTX5) which is also known as Rosenberg-Chutorian syndrome. CMTX5 is not really a classical form of CMT disease and most investigators feel the designation is inappropriate for this form of disease which is associated with peripheral nerve problems, deafness, and vision loss. Arts syndrome is associated with profound sensorineural hearing loss, hypotonia, ataxia, developmental delay, and intellectual disability predominantly in males. Manifesting females experinence much milder symptoms. In early childhood affected males will develop vision loss, peripheral neuropathy, and will have recurrent infections. As a result of the infections and the other complications of Arts syndrome affected males often do not survive past early childhood.

The PRPS2 gene is also composed of 7 exons that generate two alternatively spliced mRNAs encoding isoform 1 (321 amino acids) and isoform 2 (318 amino acids). The PRPS1L1 gene is an intronless gene located on chromosome 7p21.1 that encodes a protein of 318 amino acids. The PRPS1L1 gene is expressed exclusively in the testes and translation of the resulting mRNA begins at a non-AUG codon (ACG). Although ACG normally codes for threonine, in the PRPS1L1 mRNA this alternative start codon directs the initiator methionine for the encoded protein.

Synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP)

Synthesis of the active form of ribose. The activated form of ribose-5-phosphate is 5-phosphoribosyl-1-pyrophosphate (PRPP). Note that this reaction releases AMP. Therefore, two high energy phosphate equivalents are consumed during the reaction.

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Purine Nucleotide Biosynthesis

The major site of purine synthesis is in the liver. Synthesis of the purine nucleotides begins with PRPP and leads to the first fully formed nucleotide, inosine 5'-monophosphate (IMP). This pathway is diagrammed below. The purine base without the attached ribose moiety is hypoxanthine. The purine base is built upon the ribose by several amidotransferase and transformylation reactions. The synthesis of IMP requires five moles of ATP, two moles of glutamine, one mole of glycine, one mole of CO2, one mole of aspartate and two moles of formate. The formyl moieties are carried on tetrahydrofolate (THF) in the form of N10-formyl-THF or N5,N10-methenyl-THF.

The first reaction (1) of purine synthesis is catalyzed by an enzyme called glutamine phosphoribosylpyrophosphate amidotransferase. The activity is encoded by the PPAT gene (phosphoribosylpyrophosphate amidotransferase) located on chromsome 4q12 which is composed of 11 exons that encode a 517 amino acid protein. The activities that catalyze reactions 2, 3, and 5 are all contained in a single tri-functional enzyme encoded by the GART gene (phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase). The GART gene is located on chromsome 21q22.11 which is composed of 23 exons that generate four alternatively spliced mRNAs. Three of the mRNAs from the GART gene all encode the same protein. Reaction 4 of purine sythesis is catalyzed by phosphoribosylformylglycinamide synthase which is encoded by the PFAS gene. The PFAS gene is located on chromosome 17p13.1 which is composed of 29 exons that encode a protein of 1338 amino acids. Reactions 6 and 7 are catalyzed by a bi-functional enzyme encoded by the PAICS gene (phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase). The PAICS gene is located on chromosome 4q12 closely associated with the PPAT gene whose encoded enzyme catalyzes the first step of purine synthesis. Expression of the PPAT and PAICS genes is coordinately regulated. Reaction 8 of purine synthesis is catalyzed by adenylosuccinate lyase. Adenylosuccinate lyase is encoded by the ADSL gene located on chromosome 22q13.2 which is composed of 16 exons that generate two alternatively spliced mRNAs that encode isoform a (484 amino acids) and isoform b (425 amino acids). The last two reactions (9 and 10) are catalyzed by a bi-functional enzyme encoded by the ATIC gene (5-aminoimidazole-4-carboxyamide ribonucleotide formyltransferase, IMP cyclohydrolase). The ATIC gene is located on chromosome 2q35 which is composed of 16 exons that encode a protein of 592 amino acids.

Synthesis of purine nucleotides

Enzyme activity names:

1. glutamine phosphoribosylpyrophosphate amidotransferase (GPAT activity of the PPAT gene)

2. glycinamide ribonucleotide synthetase (GARS activity of the GART gene)

3. glycinamide ribonucleotide formyltransferase (GART activity of the GART gene)

4. phosphoribosylformylglycinamide synthase (PFAS activity of the PFAS gene)

5. aminoimidazole ribonucleotide synthetase (AIRS activity of the GART gene)

6. aminoimidazole ribonucleotide carboxylase (AIRC activity of the PAICS gene)

7. succinylaminoimidazolecarboxamide ribonucleotide synthetase (SAICAR activity of the PAICS gene)

8. adenylosuccinate lyase (ADSL activity of the ADSL gene)

9. 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT activity of the ATIC gene)

10. IMP cyclohydrolase (IMPCH activity of the ATIC gene)

De novo purine nucleotide synthesis pathway. Synthesis of the first fully formed purine nucleotide, inosine monophosphate, IMP begins with 5-phospho-α-ribosyl-1-pyrophosphate, PRPP. Through a series of reactions utilizing ATP, tetrahydrofolate (THF) derivatives, glutamine, glycine and aspartate this pathway yields IMP. The rate limiting reaction is catalyzed by glutamine PRPP amidotransferase, enzyme indicated by 1 in the Figure. The structure of the nucleobase of IMP (hypoxanthine) is shown. Place mouse over the green intermediate names to see structures.


IMP represents a branch point for purine biosynthesis, because it can be converted into either AMP or GMP through two distinct reaction pathways. The pathway leading to AMP requires energy in the form of GTP; that leading to GMP requires energy in the form of ATP. The utilization of GTP in the pathway to AMP synthesis allows the cell to control the proportions of AMP and GMP to near equivalence. The accumulation of excess GTP will lead to accelerated AMP synthesis from IMP instead, at the expense of GMP synthesis. Conversely, since the conversion of IMP to GMP requires ATP, the accumulation of excess ATP leads to accelerated synthesis of GMP over that of AMP. The two enzymes in the IMP to AMP pathway are adenylosuccinate synthetase and adenylosuccinate lyase. Adenylosuccinate synthetase is derived from the ADSS gene which is located on chromosome 1q44 and is composed of 14 that encode a 456 amino acid protein. The adenylosuccinate lyase in this pathway is the same enzyme that catalyzes reaction 8 of de novo purine biosynthesis as described above.

The two enzymes in the IMP to GMP pathway are IMP dehydrogenase (IMPDH) and GMP synthetase. Humans express two IMPDH genes identified as IMPDH1 and IMPDH2. The IMPDH1 gene is located on chromosome 7q31.3–q32 and is composed of 18 exons that generate eight alternatively spliced mRNAs encoding eight protein isoforms. The IMDPH2 gene is located on chromosome 3p21.2 and is composed of 15 exons that encode a 514 amino acid protein. Expression of IMPDH1 predominates in the retina, spleen, and resting peripheral blood mononuclear cells but like the IMPDH2 gene is also expressed in most tissues at varying levels. Regardless of the tissue, IMPDH1 is expressed constitutively at low levels. Expression of IMPDH2 is enhanced during proliferation and transformation. GMP synthetase is derived from the GMPS gene which is located on chromosome 3q24 and is composed of 18 exons that encode a 693 amino acid protein.

Synthesis of AMP and GMP from IMP

Synthesis of AMP and GMP from IMP. Following the synthesis of IMP, this nucleotide can serve as a precursor for both AMP and GMP synthesis. The direction of the pathway is controlled by the level of the respective nucleotide. When guanine nucleotide levels are high, IMP is directed to the synthesis of AMP with the opposite being the case when adenine nucleotide levels are higher. The pathway to AMP synthesis requires the enzymes adenylosuccinate synthetase and adenylosuccinate lyase. The enzymes required for the synthesis of GMP are IMP dehydrogenase 1 and GMP synthetase.


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Regulation of Purine Nucleotide Synthesis

The essential rate limiting steps in purine biosynthesis occur at the first two steps of the pathway. The synthesis of PRPP by PRPP synthetase is feed-back inhibited by purine-5'-nucleotides (predominantly AMP and GMP). Combinatorial effects of those two nucleotides are greatest, e.g., inhibition is maximal when the correct concentration of both adenine and guanine nucleotides is achieved.

The amidotransferase reaction catalyzed by PRPP amidotransferase is also feed-back inhibited allosterically by binding ATP, ADP and AMP at one inhibitory site and GTP, GDP and GMP at another. Conversely the activity of the enzyme is stimulated by PRPP.

Additionally, purine biosynthesis is regulated in the branch pathways from IMP to AMP and GMP. The accumulation of excess ATP leads to accelerated synthesis of GMP, and excess GTP leads to accelerated synthesis of AMP.

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Catabolism and Salvage of Purine Nucleotides

Catabolism of the purine nucleotides leads ultimately to the production of uric acid which is insoluble and is excreted in the urine as sodium urate crystals.

Purine nucleotide catabolism

Pathways for the catabolism of the purine nucleotides. The purine mononucleotides, AMP, GMP, IMP, and XMP are all catabolized to uric acid. Each mononucleotide is first converted to the phosphate free nucleoside form through the actions of one of several cytosolic 5'-nucleotidases. The nitrogen is removed from adenosine generating inosine by the critical enzyme, adenosine deaminase, ADA. Loss of ADA activity results in the potentialy lethal disorder, severe combined immunodeficiency, SCID. The ribose is removed from the nucleosides by purine nucleoside phosphorylase (PNP) yielding the nucleobases, hypoxanthine, xanthine, and guanine. The nitrogen is removed from guanine by guanine deaminase yielding xanthine. Hypoxanthine and xanthine are then converted to the terminal product of purine catabolis, uric acid, by the enzyme xanthine oxidase.

The synthesis of nucleotides from the purine bases and purine nucleosides takes place in a series of steps known as the salvage pathways. The free purine bases, adenine, guanine, and hypoxanthine, can be reconverted to their corresponding nucleotides by phosphoribosylation where PRPP, like in the de novo synthesis pathway, serves as the activated form of ribose-5-phosphate. Two key transferase enzymes are involved in the salvage of purines: adenosine phosphoribosyltransferase (APRT), which catalyzes the following reaction:

adenine + PRPP <——> AMP + PPi

and hypoxanthine-guanine phosphoribosyltransferase (HGPRT), which catalyzes the following reactions:

hypoxanthine + PRPP <——> IMP + PPi

guanine + PRPP <——> GMP + PPi

A critically important enzyme of purine salvage in rapidly dividing cells is adenosine deaminase (ADA) which catalyzes the deamination of adenosine to inosine. Deficiency in ADA results in the disorder called severe combined immunodeficiency, SCID (and briefly outlined below).

Purine nucleotide salvage

Salvage pathways for purine nucleotides. Salvage of the purine nucleobases, adenine, hypoxanthine, and guanine, involves several enzymes, three of which are highly clinically relevant as evidenced by the pathology associated with deficiencies in those enzymes. The critical enzymes are adenosine phosphoribosyltransferase (APRT), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and adenosine deaminase (ADA). The purine catabolic enzyme, PNP, can also function in the reverse direction (salvage) incorporating ribose into the nucleobases forming the respective nucleosides, although this latter reaction is much less significant than the reactions catalyzed by APRT and HGPRT.

The synthesis of AMP from IMP and the salvage of IMP via AMP catabolism have the net effect of deaminating aspartate to fumarate. This process has been termed the purine nucleotide cycle (see diagram below). This cycle is very important in muscle cells. Increases in muscle activity create a demand for an increase in the TCA cycle, in order to generate more NADH for the production of ATP. However, muscle lacks most of the enzymes of the major anapleurotic reactions. Muscle replenishes TCA-cycle intermediates in the form of fumarate generated by the purine nucleotide cycle.

Purine nucleotide cycle

The purine nucleotide cycle serves an important function within exercising muscle. The generation of fumarate provides skeletal muscle with its' only source of anapleurotic substrate for the TCA cycle. In order for continued operation of the cycle during exercise, muscle protein must be utilized to supply the amino nitrogen for the generation of aspartate. The generation of asparate occurs by the standard transamination reactions that interconvert amino acids with α-ketoglutarate to form glutamate and glutamate with oxaloacetate to form aspartate. Myoadenylate deaminase is the muscle-specific isoenzyme of AMP deaminase, and deficiencies in myoadenylate deaminase lead to post-exercise fatigue, cramping and myalgias.


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Clinical Significances of Purine Metabolism

Clinical problems associated with nucleotide metabolism in humans are predominantly the result of abnormal catabolism of the purines. The clinical consequences of abnormal purine metabolism range from mild to severe and even fatal disorders. Clinical manifestations of abnormal purine catabolism arise from the insolubility of the degradation byproduct, uric acid. Gout is a condition that results from the precipitation of urate as monosodium urate (MSU) or calcium pyrophosphate dihydrate (CPPD) crystals in the synovial fluid of the joints, leading to severe inflammation and arthritis. The inflammatory response is due to the crystals engaging the caspase-1-activating inflammasome resulting in the production of interleukin-1β (IL-1β) and IL-18. Most forms of gout are the result of excess purine production and consequent catabolism or to a partial deficiency in the salvage enzyme, HGPRT. Most forms of gout can be treated by administering the antimetabolite: allopurinol. This compound is a structural analog of hypoxanthine that strongly inhibits xanthine oxidase.

Two severe disorders, both quite well described, are associated with defects in purine metabolism: Lesch-Nyhan syndrome and severe combined immunodeficiency disease (SCID). Lesch-Nyhan syndrome results from the loss of a functional HGPRT gene. The disorder is inherited as a sex-linked trait, with the HGPRT gene on the X chromosome (Xq26–q27.2). Patients with this defect exhibit not only severe symptoms of gout but also a severe malfunction of the nervous system. In the most serious cases, patients resort to self-mutilation. Death usually occurs before patients reach their 20th year.

SCID refers to a group of potentially fatal disorders due to a combined loss of function of both T- and B-lymphocytes. There are at least 13 known and characterized genetic causes of SCID. The most common (45%) cause of SCID is the X-linked disorder resulting from loss of function of the common gamma (γ) chain of the T-cell receptor and other interleukin (IL) receptors. The second most common (15%) form of SCID is caused by defects in the enzyme adenosine deaminase, ADA. This is the enzyme responsible for converting adenosine to inosine in the catabolism of the purines. This deficiency selectively leads to a destruction of B and T lymphocytes, the cells that mount immune responses. In the absence of ADA, deoxyadenosine is phosphorylated to yield levels of dATP that are 50-fold higher than normal. The levels are especially high in lymphocytes, which have abundant amounts of the salvage enzymes, including nucleoside kinases. High concentrations of dATP inhibit ribonucleotide reductase (see below), thereby preventing other dNTPs from being produced. The net effect is to inhibit DNA synthesis. Since lymphocytes must be able to proliferate dramatically in response to antigenic challenge, the inability to synthesize DNA seriously impairs the immune responses, and the disease is usually fatal in infancy unless special protective measures are taken. A less severe immunodeficiency results when there is a lack of purine nucleoside phosphorylase (PNP), another purine-degradative enzyme.

One of the many glycogen storage diseases von Gierke disease also leads to excessive uric acid production. This disorder results from a deficiency in glucose 6-phosphatase activity. The increased availability of glucose-6-phosphate increases the rate of flux through the pentose phosphate pathway, yielding an elevation in the level of ribose-5-phosphate and consequently PRPP. The increases in PRPP then result in excess purine biosynthesis.

Disorders of Purine Metabolism

Disorder Defect Nature of Defect Comments
Gout PRPP synthetase
increased enzyme activity hyperuricemia
Gout HGPRTa enzyme deficiency hyperuricemia
Gout glucose-6-phosphatase enzyme deficiency hyperuricemia
Lesch-Nyhan syndrome HGPRT lack of enzyme see above
SCID ADAb lack of enzyme see above
Immunodeficiency PNPc lack of enzyme see above
Renal lithiasis APRTd lack of enzyme 2,8-dihydroxyadenine, renal lithiasis
Xanthinuria Xanthine oxidase lack of enzyme hypouricemia and xanthine renal lithiasis
von Gierke disease Glucose-6-phosphatase enzyme deficiency see above

a hypoxanthine-guanine phosphoribosyltransferase

b adenosine deaminase

c purine nucleotide phosphorylase

d adenosine phosphoribosyltransferase

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Pyrimidine Nucleotide Biosynthesis

Synthesis of the pyrimidines is less complex than that of the purines, since the base is much simpler. The first completed base is derived from one mole of glutamine, one mole of ATP and one mole of CO2 (which form carbamoyl phosphate) and one mole of aspartate. An additional mole of glutamine and ATP are required in the conversion of UTP to CTP. The pathway of pyrimidine biosynthesis is diagrammed below. The synthesis of pyrimidines differs in two significant ways from that of purines. First, the ring structure is assembled as a free base, not built upon PRPP. PRPP is added to the first fully formed pyrimidine base (orotic acid), forming orotate monophosphate (OMP), which is subsequently decarboxylated to UMP. Second, there is no branch in the pyrimidine synthesis pathway.

The carbamoyl phosphate used for pyrimidine nucleotide synthesis is derived from glutamine and bicarbonate, within the cytosol, as opposed to the urea cycle carbamoyl phosphate derived from ammonia and bicarbonate in the mitochondrion. The urea cycle reaction is catalyzed by carbamoyl phosphate synthetase I (CPS-I, CPS-1) whereas the pyrimidine nucleotide precursor is synthesized by the CPS-II (CPS-2) activity of the tri-functional rate-limiting enzyme of pyrimidine nucleotide biosynthesis. The carbamoyl phosphate that is produced by this enzyme is then condensed with aspartate in the second step of the reaction catalyzed by the aspartate transcarbamoylase (ATCase) activity of the enzyme. The third step of pyrimidine nucleotide biosynthesis is catalyzed by the dihydroorotase activity (previously referred to as carbamoyl aspartate dehydratase) of the tri-functional enzyme. The official name for this tri-functional enzyme is carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase. This enzyme is encoded by the CAD gene located on chromosome 2p22–p21 which is composed of 45 exons that encode a protein of 2225 amino acids.

Reaction catalyzed by carbamoylphosphate synthetase II

Synthesis of carbamoyl phosphate. The CPS-2 (CPS-II) activity of the trifunctional enzyme encoded by the CAD gene (carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase) utilizes glutamine as the nitrogen donor in the first step of de novo pyrimidine nucleotide synthesis. Reactions labeled as 1 and 2 in the next Figure are catalyzed by the other two activities of the CAD encoded enzyme.


Pyrimidine nucleotide synthesis

Enzyme names/activities:

1. aspartate transcarbamoylase (ATCase) activity of the CAD gene encoded enzyme

2. dihydroorotase (previously called carbamoyl aspartate dehydratase) activity of the CAD gene encoded enzyme

3. dihydroorotate dehydrogenase (DHODH gene activity)

4. orotate phosphoribosyltransferase activity of the UMPS gene

5. orotidine-5'-phosphate decarboxylase (OMP decarboxylase) activity of the UMPS gene

The activities of CPS-2 and reactions 1 and 2 are all catalyzed by the trifunctional enzyme encoded by the CAD gene

The activities of 4 and 5 are contained in a single bifunctional enzyme encoded by the uridine monophosphate synthetase (UMPS) gene

Synthesis of UMP from carbamoyl phosphate. Carbamoyl phosphate utilized in pyrimidine nucleotide synthesis differs from that synthesized in the urea cycle; it is synthesized from glutamine instead of ammonia and is synthesized in the cytosol. The reaction is catalyzed by the carbamoyl phosphate synthetase 2 (CPS-2) activity of CAD. Subsequently carbamoyl phosphate is incorporated into the pyrimidine nucleotide biosynthesis pathway through the action of the aspartate transcarbamoylase (ATCase) activity of CAD (reaction 1). The activities of CAD constitute the rate-limiting steps of pyrimidine nucleotide biosynthesis. The overal activity of the CAD encided enzyme is regulated by AMPK-mediated phosphorylation. Place mouse over green intermediate names to see structure.

Following the synthesis of dihydroorotic acid by the tri-functional CAD enzyme this compound is oxidized to orotic acid by dihydroorotate dehydrogenase (reaction 3). Dihydroorotate dehydrogenase is a mitochondrial enzyme tethered to the outer face of the inner mitochondrial membrane. This enzyme is encoded by the DHODH gene which is located on chromosome 16q22 and is composed of 11 exons that encode a protein of 395 amino acids. The last two reactions (4 and 5), which generatre the first fully formed pyrimidine nucletide (UMP), are catalyzed by the bi-functional homodimeric enzyme identified as UMP synthetase. This enzyme possesses orotate phosphoribosyltransferase activity in the N-terminal domain and OMP decarboxylase activity in the C-terminal domain. The UMPS gene is located on chromosome 3q13 and is composed of 7 exons that encode a protein of 480 amino acids.

Following the completion of UMP synthesis this nucleotide is phosphorylated twice to yield UTP (ATP is the phosphate donor). The first phosphorylation is catalyzed by uridylate kinase and the second by ubiquitous nucleoside diphosphate kinase. The synthesis of CTP occurs through the amination of UTP by the action of CTP synthase. Humans possess two distinct CTP synthase genes, CTPS1 and CTPS2. The CTPS1 gene is located on chromosome 1p34.1 and is composed of 22 exons that produce several alternatively spliced mRNAs. The CTPS2 gene is located on chromosome Xp22 and is also composed of 22 exons that undergo alternative splicing.

Uridine nucleotides are also the precursors for de novo synthesis of the thymine nucleotides (next section). The thymine nucleotides are in turn derived by de novo synthesis from dUMP or by salvage pathways from deoxyuridine or deoxythymidine.

Synthesis of CTP from UTP

Synthesis of CTP from UTP


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Synthesis of the Thymine Nucleotides

The de novo pathway to dTTP synthesis first requires the use of dUMP from the metabolism of either UDP or CDP. The dUMP is converted to dTMP by the action of thymidylate synthetase. The methyl group (recall that thymine is 5-methyl uracil) is donated by N5,N10-methylene THF, similarly to the donation of methyl groups during the biosynthesis of the purines. The unique property of the action of thymidylate synthetase is that the THF is converted to dihydrofolate (DHF), the only such reaction yielding DHF from THF. In order for the thymidylate synthetase reaction to continue, THF must be regenerated from DHF. This is accomplished through the action of dihydrofolate reductase (DHFR). THF is then converted to N5,N10-THF via the action of serine hydroxymethyl transferase. The crucial role of DHFR in thymidine nucleotide biosynthesis makes it an ideal target for chemotherapeutic agents (see below).

The thymidylate synthetase gene (symbol: TYMS) is located on chromosome 18p11.32 and is composed of 7 exons that encode a protein of 313 amino acids. A naturally occurring antisense RNA is derived by reverse transcription of the TYMS gene. This RNA is identified as rTSα. Expression of TYMS and the antisense RNA varies inversely during progression through the cell cycle. The DHFR gene is located on chromosome5q14.1 and is composed of 6 exons that generate three alternatively spliced mRNAs that encode three distinct isoforms of DHFR.

Synthesis of dTMP from dUMP

Synthesis of dTMP from dUMP

The salvage pathway to dTTP synthesis involves the enzyme thymidine kinase which can use either thymidine or deoxyuridine as substrate:

thymidine + ATP <——> TMP + ADP

deoxyuridine + ATP <——> dUMP + ADP

The activity of thymidine kinase (one of the various deoxyribonucleotide kinases) is unique in that it fluctuates with the cell cycle, rising to peak activity during the phase of DNA synthesis; it is inhibited by dTTP.

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Clinical Relevance of Tetrahydrofolate

Tetrahydrofolate (THF) is regenerated from the dihydrofolate (DHF) product of the thymidylate synthetase reaction by the action of dihydrofolate reductase (DHFR), an enzyme that requires NADPH. Cells that are unable to regenerate THF suffer defective DNA synthesis and eventual death. For this reason, as well as the fact that dTTP is utilized only in DNA, it is therapeutically possible to target rapidly proliferating cells over non-proliferating cells through the inhibition of thymidylate synthetase. Many anti-cancer drugs act directly to inhibit thymidylate synthetase, or indirectly, by inhibiting DHFR.

The class of molecules used to inhibit thymidylate synthetase is called the suicide substrates because they irreversibly inhibit the enzyme. Molecules of this class include 5-fluorouracil and 5-fluorodeoxyuridine. Both are converted within cells to 5-fluorodeoxyuridylate, FdUMP. It is this drug metabolite that inhibits thymidylate synthetase. Many DHFR inhibitors have been synthesized, including methotrexate, aminopterin, and trimethoprim. Each of these is an analog of folic acid.

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Regulation of Pyrimidine Biosynthesis

The regulation of pyrimidine synthesis occurs mainly at the first step which is catalyzed by the trifunctional enzyme encoded by the CAD gene. The ATCase activity of the enzyme is inhibited by CTP and activated by ATP. The carbamoyl synthetase activity of this complex is termed carbamoyl phosphate synthetase II (CPS-II), as opposed to CPS-I which is involved in the urea cycle. The CAD encoded enzyme is localized to the cytoplasm and the CPS-II activity utilizes glutamine as the nitrogen donor for the synthesis of carbamoyl phosphate. CPS-I of the urea cycle is localized in the mitochondria and utilizes ammonia. The CPS-II domain is activated by ATP and inhibited by UDP, UTP, dUTP, and CTP.

The role of glycine in the regulation of the CAD gene tri-functional enzyme is exerted on the ATCase activity of the complex. ATP levels also regulate pyrimidine nucleotide biosynthesis at the level of PRPP formation. An increase in the level of PRPP results in an activation of pyrimidine synthesis.

There is also regulation of OMP decarboxylase activity of the bi-functional OMP synthase enzyme. The decarboxylase activity domain is competitively inhibited by UMP and, to a lesser degree, by CMP. Finally, CTP synthase is feedback-inhibited by CTP and activated by GTP.

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Catabolism and Salvage of Pyrimidine Nucleotides

Catabolism of the pyrimidine nucleotides leads ultimately to β-alanine (when CMP and UMP are degraded) or β-aminoisobutyrate (when dTMP is degraded) and NH3 and CO2. The β-alanine and β-aminoisobutyrate serve as -NH2 donors in transamination of α-ketoglutarate to glutamate. A subsequent reaction converts the products to malonyl-CoA (which can be diverted to fatty acid synthesis) or methylmalonyl-CoA (which is converted to succinyl-CoA and can be shunted to the TCA cycle).

The salvage of pyrimidine bases has less clinical significance than that of the purines, owing to the solubility of the by-products of pyrimidine catabolism. However, as indicated above, the salvage pathway to thymidine nucleotide synthesis is especially important in the preparation for cell division. Uracil can be salvaged to form UMP through the concerted action of uridine phosphorylase and uridine kinase, as indicated:

uracil + ribose-1-phosphate <——> uridine + Pi

uridine + ATP ——> UMP + ADP

Deoxyuridine is also a substrate for uridine phosphorylase. Formation of dTMP, by salvage of dTMP requires thymine phosphorylase and the previously encountered thymidine kinase:

thymine + deoxyribose-1-phosphate <——> thymidine + Pi

thymidine + ATP ——> dTMP + ADP

The salvage of deoxycytidine is catalyzed by deoxycytidine kinase:

deoxycytidine + ATP <——> dCMP + ADP

Deoxyadenosine and deoxyguanosine are also substrates for deoxycytidine kinase, although the Km for these substrates is much higher than for deoxycytidine.

The major function of the pyrimidine nucleoside kinases is to maintain a cellular balance between the level of pyrimidine nucleosides and pyrimidine nucleoside monophosphates. However, since the overall cellular and plasma concentrations of the pyrimidine nucleosides, as well as those of ribose-1-phosphate, are low, the salvage of pyrimidines by these kinases is relatively inefficient.

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Clinical Significances of Pyrimidine Metabolism

Because the products of pyrimidine catabolism are soluble, few disorders result from excess levels of their synthesis or catabolism. Two inherited disorders affecting pyrimidine biosynthesis are the result of deficiencies in the bifunctional enzyme catalyzing the last two steps of UMP synthesis, orotate phosphoribosyltransferase and OMP decarboxylase. These deficiencies result in two types of orotic aciduria (type 1 and type 2) that cause retarded growth, and severe anemia associated with hypochromic erythrocytes and megaloblastic bone marrow, both of which are the result of the block to DNA synthesis. Leukopenia is also common in orotic acidurias. These disorders can be treated with uridine and/or cytidine, which leads to increased UMP production via the action of nucleoside kinases. The UMP then inhibits the CPS-II activity of the CAD encoded enzyme, thus attenuating orotic acid production.

Disorders of Pyrimidine Metabolism

Disorder Defective Enzyme Comments
Orotic aciduria, type 1 due to defects in both the orotate phosphoribosyltransferase and OMP decarboxylase activities of the bifunctional enzyme encoded by the UMPS gene see above for details; NO associated hyperammonemia; normal BUN measurements
Orotic aciduria, type 2 due to defects in the OMP decarboxylase activity of the bifunctional enzyme encoded by the UMPS gene see above for details; NO associated hyperammonemia; normal BUN measurements
Orotic aciduria due to OTC deficiency
(no hematologic component)
the urea cycle enzyme ornithine transcarbamoylase is deficient increased mitochondrial carbamoyl phosphate exits and augments cytoplasmic pyrimidine biosynthesis; hepatic encephalopathy; associated with hyperammonemia; is NOT associated with megaloblastic anemia since no defect in nucleotide metabolism
β-aminoisobutyric aciduria transaminase, affects urea cycle function during deamination of α-amino acids to α-keto acids clinically benign; frequent in Orientals
drug induced orotic aciduria OMP decarboxylase activity of the bifunctional enzyme encoded by the UMPS gene allopurinol and 6-azauridine treatments cause orotic acidurias without a hematologic component; their catabolic by-products inhibit the OMP decarboxylase activity of the UMPS encoded bifunctional enzyme

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Formation of Deoxyribonucleotides

The typical cell contains 5 to10 times as much RNA (mRNAs, rRNAs and tRNAs) as DNA. Therefore, the majority of nucleotide biosynthesis has as its purpose the production of rNTPs. However, because proliferating cells need to replicate their genomes, the production of dNTPs is also necessary. This process begins with the reduction of rNDPs, followed by phosphorylation to yield the dNTPs. The phosphorylation of dNDPs to dNTPs is catalyzed by the same nucleoside diphosphate kinases that phosphorylates rNDPs to rNTPs, using ATP as the phosphate donor.

Ribonucleotide reductase (RR) is a heterotetrameric enzyme that contains redox-active thiol groups for the transfer of electrons during the reduction reactions. Functional RR complexes are composed of two catalytic subunits identified as M1 (large subunit) and M2 (small subunit). Within the functional RR complex there are two copies of the M1 subunit and two copies of the M2 subunit. In addition, there is a regulatory subunit identified as M2B. The M1 protein is encoded by the RRM1 gene which is located on chromosome 11p15.5 and is composed of 20 exons generate three alternatively spliced mRNAs that encode three distinct protein isoforms. The M2 protein is encoded by the RRM2 gene which is located on chromosome 2p25–p24 and is composed of 10 exons that generate two alternatively spliced mRNAs encoding two distinct protein isoforms. The M2B protein is encoded by the RRM2B gene which is located on chromosome 8q23.1 and is composed of 9 exons that generate three alternatively spliced mRNAs encoding three distinct protein isoforms.

Following the formation of a deoxynucleotide, the oxidized thiol group in RR must be returned to its reduced state. The reduction the RR thiol goups is carried out by the thioredoxin system and the glutaredoxin system. The ultimate source of the electrons is NADPH. The thioredoxin system involves the protein identified as thioredoxin (abbreviated Trx) and the enzymes identified as thioredoxin reductases (abbreviated TrxR). The TrxR enzymes function as homodimers and contain a flavin (FAD) prosthetic group and possess a binding site for NADPH which is the terminal electron donor in the reduction of RR. The glutaredoxin system involves one of several proteins of the glutaredoxin family, the anti-oxidant peptide, glutathione (abbreviated GSH), and the enzyme glutathione reductase. Like the TrxR enzymes, functional glutathione reductase contains an FAD prosthetic group and an NADPH-binding site.

Thioredoxin is derived from the TXN gene which is located on chromosome 9q31 and is composed of 5 exons that generate two alternatively spliced mRNAs encoding two distinct cytoplasmic protein isoforms. Humans express three thioredoxin reductase genes identified as TXNRD1, TXNRD2, and TXNRD3. The TXNRD1 gene is located on chromosome 12q23.3 and is composed of 18 exons that generate seven alternatively spliced mRNAs that collectively encode five protein isoforms. All of the TXNRD1 encoded enzymes are cytoplasmic and are the principal enzymes involved in deoxynucleotide synthesis. The TXNRD2 gene is located on chromosome 22q11.21 and is composed of 19 exons that generate two alternatively spliced mRNAs encoding two different protein isoforms. The TXNRD2 encoded proteins are localized to the mitochondria and are primarily involved in scavenging reactive oxygen species in this organelle. The TXNRD3 gene is located on chromosome 3q21.3 and is composed of 16 exons that generate two alternatively spliced mRNAs encoding two distinct protein isoforms. Like the TXNRD1 encoded proteins, the TXNRD3 encoded proteins are involved in the formation of deoxynucleotides.

The glutaredoxins are a family of glutathione-dependent proteins that function in a variety of cellular redox reactions including the formation of deoxynucleotides. Humans express five genes that encode proteins containing the glutaredoxin functional domain. Four of the five proteins are called glutaredoxins, the fifth protein is the enzyme identified as prostaglandin E synthase 2 (encoded by the PTGES2 gene). The four glutaredoxin proteins are encoded by the GLRX, GLRX2, GLRX3, and GLRX5 genes. The GLRX gene is located on chromosome 5q14 and is composed of 3 exons that generate four alternatively spliced mRNAs, each of which encode the same 106 amino acid cytoplasmic protein. The GLRX encoded protein is the primary glutaredoxin involved in the formation of deoxynucleotides. The GLRX2 gene is located on 1q31.2 and is composed of 6 exons that generate four alternatively spliced mRNAs that encode three distinct protein isoforms that possess distinct subcellular patterns of localization that include the mitochondria, cytosol, and nucleus. The GLRX3 gene is located on chromosome 10q26 and is composed of 13 exons that generate three alternatively spliced mRNAs encoding two distinct protein isoforms. The primary function of the GLRX3 encoded proteins is in the regulation of the function of a specific PKC isoform (PKCθ). The GLRX5 gene is located on chromosome 14q32.13 and is composed of 2 exons that encode a 157 amino acid precursor protein. The GLRX5 encoded protein is localized to the mitochondria where it functions in the formation of iron-sulfur centers in complexes of the oxidative phosphorylation pathway. The glutathione reductase gene (symbol: GSR) is located on chromosome 8p21.1 and is composed of 13 exons that generate four alternatively spliced mRNAs that encode four distinct mitochondrial protein isoforms.

Ribonucleotide reductase reactions

Ribonucleotide reductase reactions. The primary pathway for the synthesis of the deoxynucleotides involves the association of the redox reactions of thioredoxin and thioredoxin reductase. However, the glutaredoxin, glutathione, and glutathione reductase pathway does serve as an important component of the overall deoxynucleotide synthesis process.

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Regulation of dNTP Formation

Ribonucleotide reductase is the only enzyme used in the generation of all the deoxyribonucleotides. Therefore, its activity and substrate specificity must be tightly regulated to ensure balanced production of all four of the dNTPs required for DNA replication. Such regulation occurs by binding of nucleoside triphosphate effectors to either the activity sites or the specificity sites of the enzyme complex. The activity sites bind either ATP or dATP with low affinity, whereas the specificity sites bind ATP, dATP, dGTP, or dTTP with high affinity. The binding of ATP at activity sites leads to increased enzyme activity, while the binding of dATP inhibits the enzyme. The binding of nucleotides at specificity sites effectively allows the enzyme to detect the relative abundance of the four dNTPs and to adjust its affinity for the less abundant dNTPs, in order to achieve a balance of production. thioredoxin reductase and glutathione reductase respectively.

The ribonucleotide reductase enzyme is a heterotetrameric enzyme composed of two different subunits. The large common subunit is encoded by the RRM1 gene. There are two genes encoding the small subunit of functional ribonucleotide reductase. One of the genes (RRM2) is constitutively expressed, the other (RRM2B) is induced via the transcription factor, p53. The RRM1 gene is located on chromosome 11p15.5 and is composed of 19 exons that encode a protein of 792 amino acids. The RRM1 gene resides in the region of chromosome 11 that is deleted in Beckwith-Wiedemann syndrome. The RRM2 gene is located on chromosome 2p25-p24 and is composed of 10 exons that generate two alternatively spliced mRNA generating isoform 1 (449 amino acids) and isoform 2 (389 amino acids). The RRM2B gene is located on chromosome 8q23.1 and is composed of 9 exons that yield several alternatively spliced mRNAs.

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Interconversion of the Nucleotides

During the catabolism of nucleic acids, nucleoside mono- and diphosphates are released. The nucleosides do not accumulate to any significant degree, owing to the action of nucleoside kinases. These include both nucleoside monophosphate (NMP) kinases and nucleoside diphosphate (NDP) kinases. The NMP kinases catalyze ATP-dependent reactions of the type:

(d)NMP + ATP <——> (d)NDP + ADP

There are four classes of NMP kinases that catalyze, respectively, the phosphorylation of:

1. AMP and dAMP; this kinase is known as adenylate kinase.

2. GMP and dGMP.

3. CMP, UMP and dCMP.

4. dTMP.

The enzyme adenylate kinase is important for ensuring adequate levels of energy in cells such as liver and muscle. The predominant reaction catalyzed by adenylate kinase is:

2ADP <——> AMP + ATP

The NDP kinases catalyze reaction of the type:

N1TP + N2DP <——> N1DP + N2TP

N1 can represent a purine ribo– or deoxyribonucleotide; N2 a pyrimidine ribo– or deoxyribonucleotide. The activity of the NDP kinases can range from 10 to 100 times higher than that of the NMP kinases. This difference in activity maintains a relatively high intracellular level of (d)NTPs relative to that of (d)NDPs. Unlike the substrate specificity seen for the NMP kinases, the NDP kinases recognize a wide spectrum of (d)NDPs and (d)NTPs.

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Last modified: July 20, 2016