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Biochemistry · Metabolic pathway

Pyrimidine synthesis !

Pyrimidines are built the other way round from purines: ring first (orotate), sugar second. UMP, CTP and dTMP come from there.

Updated 2026-11-01

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Key facts

  • Ring first, sugar second: orotate joins PRPP at step 5.
  • CPS-II (cytosol, uses glutamine) is the rate-limiting step in humans; UTP inhibits it and PRPP and ATP activate it. Not to be confused with CPS-I (mitochondria, ammonia, urea cycle).
  • Three proteins do almost everything: CAD (steps 1–3), DHODH (step 4, in mitochondria) and UMP synthase (steps 5–6).
  • Deoxyribonucleotides come from ribonucleotide reductase (diphosphates); dTMP forms by methylation of dUMP by thymidylate synthase.
  • Ring atoms: aspartate (N1, C4, C5, C6), CO₂ (C2) and glutamine (N3).

In detail

  • CTP is made from UTP by CTP synthetase, which takes nitrogen from glutamine.
  • Steps 9–10: NDP kinase converts dUDP to dUTP and dUTPase cuts it to dUMP, which keeps uracil out of DNA.
  • Another source of dUMP is deamination of dCMP (dCMP deaminase).
  • DHODH hands its electrons to ubiquinone, linking pyrimidine synthesis with the respiratory chain.
  • Thymidylate synthase oxidises folate to DHF; DHFR returns it to THF. That is why methotrexate also stops thymidine synthesis.
Also called
Orotate pathway

Regulation

  • CPS-II: UTP (−), PRPP and ATP (+); phosphorylation by MAP kinases activates it when the cell grows.
  • Ribonucleotide reductase is regulated by dATP (off) and ATP (on) and chooses its substrate according to the bound dNTP.
  • In bacteria control lies at aspartate transcarbamoylase (inhibited by CTP); in humans, at CPS-II.

The steps, one by one

  1. Step 1 · Carbamoyl phosphate synthetase II (CAD) irreversible Glutamine + CO₂ + ATP → Carbamoyl phosphate + Glutamate + ADP + phosphate (Pi)Rate-limiting step in the cytosol: bicarbonate and glutamine nitrogen form carbamoyl phosphate using 2 ATP.If it fails: CAD deficiency causes an epileptic encephalopathy that improves with uridine.
  2. Step 2 · Aspartate transcarbamoylase (CAD domain) irreversible Carbamoyl phosphate + Aspartate → Carbamoyl aspartate + phosphate (Pi)Joins aspartate to carbamoyl phosphate; most of the ring atoms enter here.If it fails: It is part of CAD; its defects appear as CAD deficiency.
  3. Step 3 · Dihydroorotase (CAD domain) Carbamoyl aspartate ⇌ Dihydroorotate + H₂OCloses the six-membered ring by removing a water molecule.If it fails: It is part of CAD; its defects appear as CAD deficiency.
  4. Step 4 · Dihydroorotate dehydrogenase (DHODH) irreversible Dihydroorotate + Ubiquinone (CoQ) → Orotic acid + Ubiquinol (CoQH₂)The only mitochondrial step: oxidises the ring to orotate and passes electrons to ubiquinone.If it fails: DHODH variants cause Miller syndrome (postaxial acrofacial dysostosis).
  5. Step 5 · UMP synthase: orotate phosphoribosyltransferase Orotic acid + PRPP ⇌ Orotidine monophosphate (OMP) + pyrophosphateThe finished ring receives ribose phosphate from PRPP and becomes a nucleotide (OMP).If it fails: Its deficiency causes hereditary orotic aciduria. Hereditary orotic aciduria →
  6. Step 6 · UMP synthase: OMP decarboxylase irreversible Orotidine monophosphate (OMP) → Uridine monophosphate (UMP) + CO₂Removes the orotate carboxyl and forms UMP, the first complete pyrimidine.If it fails: Its deficiency causes hereditary orotic aciduria. Hereditary orotic aciduria →
  7. Step 7 · UMP/CMP kinase Uridine monophosphate (UMP) + ATP ⇌ Uridine diphosphate (UDP) + ADPAdds a phosphate, giving UDP, the substrate of ribonucleotide reductase.If it fails: It also phosphorylates analogues such as gemcitabine monophosphate.
  8. Step 8 · Ribonucleotide reductase irreversible Uridine diphosphate (UDP) → Deoxyuridine diphosphate (dUDP) + H₂ORemoves the oxygen at C2' of the ribose: the ribonucleotide becomes a deoxyribonucleotide.If it fails: dATP that builds up in ADA deficiency switches it off and kills lymphocytes. Adenosine deaminase deficiency (ADA-SCID) →
  9. Step 9 · Nucleoside diphosphate kinase Deoxyuridine diphosphate (dUDP) + ATP ⇌ Deoxyuridine triphosphate (dUTP) + ADPMoves a phosphate from ATP to dUDP, forming dUTP.If it fails: An abundant, non-specific enzyme; no known disease from its loss.
  10. Step 10 · dUTPase irreversible Deoxyuridine triphosphate (dUTP) + H₂O → Deoxyuridine monophosphate (dUMP) + pyrophosphateCuts dUTP to dUMP, keeping dUTP low so DNA polymerase does not put uracil into DNA.If it fails: When thymidylate synthase is blocked (5-FU), uracil enters DNA and breaks during repair.
  11. Step 11 · Thymidylate synthase irreversible Deoxyuridine monophosphate (dUMP) + N⁵,N¹⁰-Methylene-tetrahydrofolate → dTMP + Dihydrofolate (DHF)Puts a methyl on C5 of uracil: dUMP becomes dTMP and the folate is left oxidised (DHF).If it fails: Lack of folate or B12 slows this step: megaloblastic anaemia.

Clinical notes

  • Hereditary orotic aciduria: UMP synthase fails, orotate builds up and a megaloblastic anaemia appears that does not respond to B12 or folate.
  • In OTC deficiency (urea cycle) mitochondrial carbamoyl phosphate spills into the cytosol and orotic acid also rises, but with hyperammonaemia.
  • Drugs: leflunomide (DHODH), 5-fluorouracil (thymidylate synthase), methotrexate (DHFR) and hydroxyurea (ribonucleotide reductase).

Test yourself

Think of the answer, then tap the question to see it.

How do CPS-I and CPS-II differ?

CPS-I is mitochondrial, uses ammonia and is activated by N-acetylglutamate (urea); CPS-II is cytosolic, uses glutamine and is inhibited by UTP (pyrimidines).

Which lab finding separates OTC deficiency from hereditary orotic aciduria?

Both raise urinary orotic acid, but only OTC deficiency comes with hyperammonaemia.

Which enzyme of the pathway sits in mitochondria, and which drug blocks it?

Dihydroorotate dehydrogenase; leflunomide (through its metabolite teriflunomide) blocks it.

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Educational information, not medical advice. For any symptom, see a health professional.

Sources: Pyrimidine biosynthesis (Reactome, 2026); Interconversion of nucleotide di- and triphosphates (Reactome, 2026); CAD: Multifunctional protein CAD (human) (UniProt, 2026); Textbook of Biochemistry for Medical Students, 6th ed. (Jaypee Brothers Medical Publishers (Vasudevan DM, Sreekumari S, Vaidyanathan K), 2011).

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