Purine and pyrimidine 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.
De novo synthesis of purine nucleotides
- Pathway: ribose 5-phosphate → phosphoribosyl pyrophosphate (PRPP) → (10 steps) → inosine-5'-monophosphate (IMP) → (2 steps each) → 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
- PRPP synthesis
- Key enzyme (committed step of the de novo synthesis): glutamine PRPP amidotransferase
- Reaction: PRPP + glutamine → 5-phosphoribosylamine (PRA), glutamate, PPi
- 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)
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
- Clinical significance: IMP dehydrogenase is inhibited by mycophenolic acid
- Second reaction step: XMP + ATP + glutamine → guanosine-5'-monophosphate (GMP) + AMP + PPi + 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!
Inhibitors of de novo purine synthesis
6-mercaptopurine (6-MP): inhibit conversion of PRPP to IMP
- Prodrug of 6-MP: azathioprine
- Mycophenolate and ribavirin: inhibit inosine monophosphate dehydrogenase
- Hydroxyurea: inhibits both de novo purine and pyrimidine synthesis via inhibition of ribonucleotide reductase
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 PRPP 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 !
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 nucleotides 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
An overproduction (e.g., due to excessive purine-rich diet) or underexcretion (most common) of uric acid leads to hyperuricemia and predisposes to the join deposition of monosodium urate crystal, which causes gout.
Excessive alcohol is a common cause of hyperuricemia for multiple reasons, including: increasing purine nucleotide degradation during ethanol catabolism, being consumed in drinks with high amounts of purine (e.g., beer), possibly inhibiting the xanthine dehydrogenase, and inhibiting the renal excretion of urate by promoting lactic acid, dehydration, and possible ketoacidosis.
Like purine nucleotides, 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).
Pyrimidine nucleotides include the bases uracil, cytosine, and thymine, e.g. in the nucleoside triphosphates CTP (cytidine triphosphate), dTTP (deoxythymidine triphosphate) and UTP (uridine triphosphate). 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:
- Dihydroorotate dehydrogenase (located in the inner mitochondrial membrane)
- UMP synthase (located in the cytosol)
- Key reaction: aspartate + carbamoyl phosphate → carbamoyl aspartic acid + Pi
Principle of synthesis:
Glutamine + HCO3- + 2 ATP + H2O → carbamoyl phosphate + glutamate + 2 ADP + Pi
Carbamoyl phosphate + aspartate→ carbamoyl aspartate + Pi
Carbamoyl aspartic acid → → (in 2 steps to) orotic acid
Orotic acid + PRPP → → (in 2 steps to) UMP
Phase 2: synthesis of UTP and CTP
- Pathway: UMP → UDP → UTP → CTP
- Different kinases
- CTP synthetase
Phase 3: synthesis of thymine-containing deoxyribonucleotides
Description: dTMP (thymidylate) formation from dUMP through methylation
- dUMP formation: UDP reduction to dUDP by → 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
- Subsequently: phosphorylation of dTMP to dTDP and dTTP by specific kinases using ATP
Inhibitors of pyrimidine synthesis
- Methotrexate, trimethoprim, pyrimethamine: inhibit dihydrofolate reductase → ↓ dTMP in humans, bacteria, and protozoa, respectively
- Leflunomide: inhibits dihydroorotate dehydrogenase
- 5-fluorouracil (5-FU): inhibit thymidylate synthase (→ ↓ dTMP) via formation of 5-F-dUMP
- Hydroxyurea: inhibits both purine and pyrimidine synthesis via inhibition of ribonucleotide reductase
Recovery of pyrimidine nucleosides
Degradation of pyrimidine nucleotides
- 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
- Reaction steps in UMP degradation
Reaction steps in dTMP degradation
- dTMP → thymidine → thymine → dihydrothymine → β-ureidoisobutyric acid → β-aminoisobutyric acid
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
- Regeneration of SH groups of ribonucleotide reductase by thioredoxin
- Thioredoxin also contains SH groups, which are oxidized (forming a disulfide bridge).
- Regeneration of SH groups of thioredoxin by thioredoxin reductase using FADH2, which in turn is oxidized to FAD
- FAD is reduced to FADH2 by NADPH+H+, with simultaneous generation of NADP+
- NADPH+H+ is produced in the pentose phosphate pathway
The reducing equivalents for the reduction of ribonucleotides to deoxyribonucleotides are provided by NADPH+H+.