Purines and pyrimidines

Purines and pyrimidines are fundamental components of nucleotides in DNA and RNA and are essential for the storage of information in the cell. They also serve as a basic framework for coenzymes and are involved in numerous enzymatic processes. Alterations in purine or pyrimidine metabolism can have a variety of consequences. For example, disorders of purine metabolism lead to increased amounts of uric acid in blood and can result in gout. Nucleotide synthesis inhibitors are used in tumor therapy; ribonucleotide reductase inhibitor, for instance, inhibits DNA replication in highly proliferative tumor cells by depriving the building blocks of DNA.

Purine nucleotides include the bases adenine and guanine. Purine nucleotides can be newly synthesized (de novo synthesis) or recovered from degradation products (salvage pathway).

De novo synthesis of purine nucleotides

• Pathway: Ribose 5-phosphate → phosphoribosyl pyrophosphate (PRPP) → inosine-5'-monophosphate (IMP) → adenosine-5'-monophosphate (AMP) OR guanosine-5'-monophosphate (GMP)
• Nitrogen or carbon donors: 2 glutamines; , glycine, aspartate, hydrogen carbonate (HCO₃^-), 2 tetrahydrofolic acids (THF)
• Energy donators: ATP and GTP

Phases

• PRPP synthesis
  • Reaction: ribose 5-phosphate + ATP → PRPP + AMP
  • Enzyme: PRPP synthetase; (ribose-phosphate pyrophosphokinase)
• IMP synthesis
  • Key enzyme (committed step of the de novo synthesis): glutamine PRPP amidotransferase
  • Reaction: PRPP + glutamine → 5-phosphoribosylamine (PRA), glutamate, PP_i
    • Four out of 10 steps require ATP hydrolysis
    • Two out of the 10 steps require THF as a cofactor
  • Regulation
    • Allosteric activation by PRPP
    • Allosteric inhibition by AMP, GMP, and IMP
• AMP and GMP synthesis
  • AMP synthesis: oxygen atom at C6 atom of IMP exchanged by an amino group (NH₂ group)
    • First reaction step: IMP + aspartate + GTP → adenylosuccinate + GDP + P_i
    • Enzyme: adenylosuccinate synthetase
    • Second reaction step: adenylosuccinate → adenosine-5'-monophosphate (AMP) + fumarate
    • Enzyme: adenylosuccinate lyase
  • GMP synthesis: attachment of an amino group to the C2 atom
    • First reaction step: IMP + H₂O + NAD⁺ → xanthosine monophosphate (XMP) + NADH + H⁺
    • Enzyme: IMP dehydrogenase
    • Second reaction step: XMP + ATP + glutamine → guanosine-5'-monophosphate (GMP) + AMP + PP_i + glutamate
    • Enzyme: guanylate synthetase (xanthylate aminase)
• Kinases phosphorylate AMP and GMP: yield ATP and GTP, respectively.

GTP is involved in the synthesis of IMP to AMP, while ATP is involved in the synthesis of IMP to GMP!

Purine synthesis requires the 2 THF, which are reduced from dihydrofolate by dihydrofolate reductase.

Inhibitors of de novo purine synthesis

• 6-mercaptopurine (6-MP) and its prodrug azathioprine: inhibit the conversion of PRPP to IMP
• Mycophenolate and ribavirin: inhibit inosine monophosphate dehydrogenase

Purine salvage pathway

Free purine bases can be directly attached to PRPP to yield purine nucleotides. This purine nucleotide synthesis pathway is associated with significantly less energy consumption than de novo synthesis.

• Description: recycling of the purine bases adenine, guanine, and hypoxanthine
• Substrate: PRPP with adenine or with guanine and hypoxanthine
• Product: AMP or GMP and IMP
• Enzymes: adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
• Regulation: inhibition of APRT by adenine nucleotides , inhibition of HGPRT by IMP and GMP

HGPRT deficiency leads to Lesch-Nyhan syndrome!

Purine nucleotide degradation

Purine nucleotides are degraded via reaction steps that are different than those used for assembly. Because the purine ring system cannot be enzymatically cleaved in humans, purine is metabolized into uric acid and excreted in urine as urate anion.

• Description: degradation of purine nucleotides to the respective nucleosides with subsequent oxidation of the xanthine that is formed to uric acid
• Reaction steps in AMP degradation: AMP → adenosine → inosine → hypoxanthine → xanthine → uric acid
• Reaction steps in GMP degradation: GMP → guanosine→ guanine→ xanthine → uric acid
• Reaction steps in XMP degradation: XMP → xanthosine → xanthine → uric acid
• Important enzymes
  • Adenosine deaminase: catalyzes the deamination of adenosine to inosine
  • Xanthine oxidase (xanthine dehydrogenase): catalyzes the conversion of hypoxanthine to xanthine and xanthine to uric acid
  • Guanine deaminase: catalyzes the deamination of guanine to xanthine

Adenosine deaminase deficiency leads to SCID!

An overproduction (e.g., due to excessive purine-rich diet) or underexcretion (most common) of uric acid leads to hyperuricemia and predisposes to the joint deposition of monosodium urate crystal, which causes gout.

Excessive alcohol consumption is a common cause of hyperuricemia for multiple reasons, including: increased purine nucleotide degradation during ethanol catabolism, high amounts of purines in alcoholic drinks (e.g., beer), and inhibition of the renal excretion of urate (promoted by increased lactic acid blood levels, dehydration, and possible ketoacidosis).

Uric acid resulting from purine degradation should not be confused with urea resulting from nitrogen of amino acid degradation.

References:^[1]

Pyrimidine nucleotides are also newly synthesized or recovered. However, in contrast to de novo synthesis of purine nucleotides, the basic ring structure in the de novo synthesis of pyrimidine nucleotides is synthesized first and then bound to activated ribose phosphate (i.e.., PRPP).

De novo synthesis of pyrimidine nucleotides

• Pyrimidine nucleotides consist of the following bases:
  • Uracil (e.g., uridine triphosphate, UTP)
  • Cytosine (e.g., cytidine triphosphate, CTP)
  • Thymine (e.g., deoxythymidine triphosphate, dTTP)
• In pyrimidine nucleotide synthesis, uridine monophosphate (UMP) is initially formed, which can be phosphorylated to UDP and UTP. CTP can then be synthesized from UTP.
• For the synthesis of thymine-containing deoxyribonucleotides, additional reaction steps are required: First, deoxy-UMP (dUMP) is formed and is then methylated to dTMP (deoxythymidine monophosphate), which is catalyzed by thymidylate synthase.

Phase 1: synthesis pathway from aspartate and cytosolic carbamoyl phosphate to UMP

• Pathway: synthesis of carbamoyl phosphate from glutamine and bicarbonate → addition of aspartate yields carbamoyl aspartate → dihydroorotate → orotic acid → transfer to PRPP yields OMP (orotidine monophosphate) → UMP (uridine monophosphate) + CO₂
• Nitrogen or carbon donors: glutamine, bicarbonate (HCO₃^-), aspartate
• Involved enzyme complexes
  • CAD enzyme (located in the cytosol) contains three domains each with their own enzyme activity:
    • Glutamine-dependent carbamoyl phosphate synthetase 2 (CPS2): rate-limiting enzyme for de novo synthesis of pyrimidine nucleotides in the cytosol of most human cells
    • Aspartate carbamoyltransferase
    • Dihydroorotase
  • Dihydroorotate dehydrogenase (located in the inner mitochondrial membrane)
  • UMP synthase (located in the cytosol)
• Key reaction: aspartate + carbamoyl phosphate → carbamoyl aspartic acid + P_i
• Regulation
  • CPS2 activity of the CAD enzyme
    • Activation: PRPP and ATP
    • Inhibition: UTP
  • Inhibition of the dihydroorotase activity of the CAD enzyme and dihydroorotate dehydrogenase by orotic acid

Principle of synthesis:
- Glutamine + HCO₃^- + 2 ATP + H₂O → carbamoyl phosphate + glutamate + 2 ADP + P_i
- Carbamoyl phosphate + aspartate→ carbamoyl aspartate + P_i
- Carbamoyl aspartic acid → → (in 2 steps to) orotic acid
- Orotic acid + PRPP → → (in 2 steps to) UMP

UMP synthase deficiency leads to orotic aciduria characterized by megaloblastic anemia and orotic acid crystalluria!

The cytosolic carbamoyl phosphate synthetase 2 of pyrimidine synthesis should not be mistaken for the mitochondrial carbamoyl phosphate synthetase 1 of the urea cycle.

To remember that the CPS2 enzyme is located in the cytosol, think of “CyTWOsol”.

Phase 2: synthesis of UTP and CTP

• Pathway: UMP → UDP → UTP → CTP
• Enzymes:
  • Different kinases
  • CTP synthetase

Phase 3: synthesis of thymine-containing deoxyribonucleotides

• Description:
  • dTMP (thymidylate) formation from dUMP through methylation
  • UDP is reduced to dUDP by ribonucleotide reductase → phosphorylation to dUTP → dissociation of pyrophosphate → dUMP
• Enzyme: thymidylate synthase
• Reaction: dUMP + N⁵, N¹⁰-methylene tetrahydrofolate (THF) → dTMP + dihydrofolic acid
• Cofactor:
  • N⁵, N¹⁰-methylene THF (folic acid derivative) as a methyl group carrier
  • Regeneration of N⁵, N¹⁰-methylene THF:
    • Dihydrofolic acid resulting from dTMP synthesis is converted to THF by dihydrofolate reductase.
    • THF is regenerated to form N⁵, N¹⁰-methylene THF.
• Subsequently: phosphorylation of dTMP to dTDP and dTTP by specific kinases using ATP

The synthesis rate of thymine-containing deoxyribonucleotides depends on the folic acid supply!

Methotrexate and 5-fluorouracil both inhibit DNA synthesis by interfering with the formation of thymine-containing deoxyribonucleotides!

Inhibitors of pyrimidine synthesis

• Leflunomide: inhibits dihydroorotate dehydrogenase
• 5-fluorouracil (5-FU) and its prodrug capecitabine inhibit thymidylate synthase, leading to decreased dTMP via formation of 5-F-dUMP.

Inhibitors of pyrimidine and purine synthesis

• Methotrexate, trimethoprim, pyrimethamine: inhibit dihydrofolate reductase, leading to decreased dTMP in humans, bacteria, and protozoa, respectively
• Hydroxyurea: inhibits both purine and pyrimidine synthesis via inhibition of ribonucleotide reductase

Recovery of pyrimidine nucleosides

Pyrimidine nucleosides can be converted to pyrimidine nucleotides by kinases using ATP. Free pyrimidine bases without sugar residues cannot be recovered.

Degradation of pyrimidine nucleotides

In contrast to purine nucleotides, pyrimidine nucleotides can be completely degraded and used for energy generation.

• Pathway: Pyrimidine nucleotides (CMP, UMP, dTMP) → pyrimidine nucleosides → cleavage of the sugar residue yields free bases → cleavage of pyrimidine ring → β-alanine or β-aminoisobutyric acid → further conversion to malonyl-CoA or methylmalonyl-CoA
• Location: cytoplasm (especially in hepatic and renal cells)
• Reaction steps in CMP degradation
  • CMP → cytidine → uridine → uracil → dihydrouracil → 3-ureidopropionic acid → β-alanine
  • CMP can also be converted to UMP and further degraded.
• Reaction steps in UMP degradation
  • UMP → uridine → uracil → dihydrouracil → 3-ureidopropionic acid → β-alanine
• Reaction steps in dTMP degradation
  • dTMP → thymidine → thymine → dihydrothymine → β-ureidoisobutyric acid → β-aminoisobutyric acid

References:^[2]

Deoxyribonucleotides containing the purine bases adenine and guanine and the pyrimidine bases cytosine and thymine are required for DNA synthesis. Except for thymine-containing deoxyribonucleotides, the other dNTPs (deoxyribonucleoside triphosphates) are synthesized by the reduction of ribonucleotides (via ribonucleotide reductase).

• Description: reduction of ribonucleotides to deoxyribonucleotides (dATP, dGTP, dCTP as well as dUMP as a precursor of dTTP)
• Reaction: NDP (ribonucleoside diphosphate) + NADPH+H⁺ → dNDP + NADP⁺ + H₂O
• Enzyme: ribonucleotide reductase
• Further enzymes and cofactors: thioredoxin , thioredoxin reductase with the cofactor FAD, NADPH+H⁺
• Regulation: complex allosteric regulation of the activity and substrate specificity of ribonucleotide reductase, e.g. activation by ATP and inhibition by dATP
• Reaction mechanism: To reduce NDPs, a pair of cysteine SH groups of ribonucleotide reductase are oxidized and thereby form a disulfide bridge (-S–S‑).
• Additional reactions: to regenerate ribonucleotide reductase
  1. Regeneration of SH groups of ribonucleotide reductase by thioredoxin
  2. Thioredoxin also contains SH groups, which are oxidized (forming a disulfide bridge).
  3. Regeneration of SH groups of thioredoxin by thioredoxin reductase using FADH₂, which in turn is oxidized to FAD
  4. FAD is reduced to FADH₂ by NADPH+H⁺, with simultaneous generation of NADP⁺
  5. NADPH+H⁺ is produced in the pentose phosphate pathway

The reducing equivalents for the reduction of ribonucleotides to deoxyribonucleotides are provided by NADPH+H⁺.

Hydroxyurea prevents nucleotide synthesis (thus decreasing DNA synthesis) by inhibiting ribonucleotide reductase.

• Lesch-Nyhan syndrome
• Severe combined immunodeficiency
• Gout and hyperuricemia
• Orotic aciduria
• Enzymes of purine and pyrimidine metabolism are the targets of various antineoplastic, antibiotic, and immunosuppressive drugs.
  • Both purine and pyrimidine synthesis: hydroxyurea
  • Purine synthesis
    • 6-Mercaptopurine, azathioprine
    • Mycophenolate mofetil
    • Ribavirin
  • Pyrimidine synthesis
    • Methotrexate
    • Trimethoprim
    • Pyrimethamine
    • Leflunomide
    • 5-Fluorouracil