Pyrimidine De Novo Synthesis (through to UMP, then CTP/dTMP)

1. 1

   Glutamine + bicarbonate → carbamoyl phosphate

   CPS-II domain of carbamoyl-phosphate synthetase II (CPS-II) 6.3.5.5 ST-0224 Irreversible rate-limiting ATP H2O Pi Bicarbonate/CO2
   In Glutamine HCO3-

   Out Carbamoyl phosphate
   › Notes

   The CPS-II domain of carbamoyl-phosphate synthetase II (CPS-II) catalyzes glutamine + HCO3− + 2 ATP + H2O → carbamoyl phosphate + glutamate + 2 ADP + Pi. One ATP activates bicarbonate and a second phosphorylates carbamate; ammonia generated in the glutaminase domain is channeled internally. This cytosolic reaction is the committed, rate-limiting, and effectively irreversible step of mammalian pyrimidine de novo synthesis; it must not be confused with mitochondrial CPS-I of the urea cycle.

2. 2

   Carbamoyl phosphate → carbamoyl aspartate

   Aspartate transcarbamylase (ATCase) 2.1.3.2 ST-0225 Irreversible/directional Pi
   In Carbamoyl phosphate

   Out Carbamoyl aspartate
   › Notes

   Aspartate transcarbamylase (ATCase) catalyzes carbamoyl phosphate + aspartate → carbamoyl aspartate + Pi. In mammals this activity is the second domain of CAD and does not consume ATP directly.

3. 3

   Carbamoyl aspartate → dihydroorotate

   Dihydroorotase 3.5.2.3 ST-0226 Irreversible/directional H2O
   In Carbamoyl aspartate

   Out Dihydroorotate
   › Notes

   Dihydroorotase catalyzes intramolecular cyclization/dehydration: carbamoyl aspartate ⇌ L-dihydroorotate + H2O. This zinc-dependent enzyme is the third CAD activity.

4. 4

   Dihydroorotate → orotate

   Dihydroorotate dehydrogenase (DHODH) 1.3.5.2 ST-0227 Irreversible/directional Ubiquinone (CoQ)
   In Dihydroorotate

   Out Orotate
   › Notes

   DHODH catalyzes L-dihydroorotate + ubiquinone (CoQ) → orotate + ubiquinol (CoQH2). Flavin mononucleotide (FMN) is the redox cofactor; mitochondrial electron transport regenerates oxidized CoQ. This is the only mitochondrial-associated step of the pathway.

5. 5

   Orotate → orotidine 5′-monophosphate (OMP)

   Orotate phosphoribosyltransferase (UMP synthase domain 1) 2.4.2.10 ST-0228 Irreversible PPi PRPP
   In Orotate

   Out Orotidine 5'-monophosphate (OMP)
   › Notes

   Orotate phosphoribosyltransferase (OPRT), the first activity of bifunctional UMP synthase, catalyzes orotate + PRPP → OMP + PPi. Mg2+ is required, and PPi hydrolysis makes the step effectively irreversible.

6. 6

   OMP → UMP

   OMP decarboxylase (UMP synthase domain 2) 4.1.1.23 ST-0229 Irreversible/directional
   In Orotidine 5'-monophosphate (OMP)

   Out UMP
   › Notes

   OMP decarboxylase, the second UMP synthase activity, catalyzes OMP → UMP + CO2. This is one of the most proficient known enzymatic decarboxylations and commits the incorporated ribose nucleotide to the uracil nucleotide pool.

7. 7

   UMP → UDP

   UMP/CMP kinase ST-0230 Irreversible/directional ATP
   In UMP

   Out UDP
   › Notes

   UMP/CMP kinase catalyzes UMP + ATP → UDP + ADP. Mg2+ is required; this is the first phosphorylation needed to build the UTP pool.

8. 8

   UDP → UTP

   Nucleoside diphosphate kinase (NDPK/NME) 2.7.4.6 ST-0231 Irreversible/directional ATP UTP
   In UDP

   Out UTP
   › Notes

   Nucleoside diphosphate kinase (NDPK/NME) catalyzes UDP + ATP (or another NTP) ⇌ UTP + ADP (or the corresponding NDP). A phosphohistidine enzyme intermediate mediates phosphoryl transfer; the reaction is near equilibrium.

9. 9

   UTP → CTP

   CTP synthetase 6.3.4.2 ST-0232 Irreversible/directional ATP UTP H2O Pi
   In UTP

   Out CTP
   › Notes

   CTP synthetase catalyzes UTP + glutamine + ATP + H2O → CTP + glutamate + ADP + Pi. ATP activates UTP, and ammonia produced by the glutaminase domain is transferred to the synthase domain; this is the committed and regulatory step of CTP formation.

10. 10

    UDP → dUDP (route to dUMP)

    Ribonucleotide reductase (RNR) 1.17.4.1 ST-0233 Irreversible/directional
    In UDP

    Out dUDP
    › Notes

    Ribonucleotide reductase (RNR) catalyzes UDP + reducing equivalents → dUDP + oxidized electron-transfer proteins. In humans the reducing equivalents are supplied principally by NADPH through thioredoxin reductase and thioredoxin (or through glutaredoxin/glutathione); RNR is covered mechanistically in the deoxyribonucleotide section.

11. 11

    dUDP → dUTP → dUMP

    ↪ dUTP → dUMP

    Nucleoside diphosphate kinase (NDPK/NME) 2.7.4.6 ST-0234 Irreversible/directional ATP H2O PPi
    In dUDP

    Out dUTP
    › Notes

    NDPK can phosphorylate dUDP + ATP ⇌ dUTP + ADP, and dUTPase then catalyzes dUTP + H2O → dUMP + PPi. dUTPase is essential because it both supplies dUMP and keeps dUTP low enough to prevent uracil incorporation into DNA.

12. 12

    dCMP → dUMP (alternative route)

    dCMP deaminase 3.5.4.12 ST-0235 Irreversible/directional H2O
    In dCMP

    Out dUMP
    › Notes

    dCMP deaminase catalyzes dCMP + H2O → dUMP + NH3. This allosterically regulated reaction provides another major dUMP source and contributes to balanced dCTP/dTTP pools.

13. 13

    dUMP → dTMP

    Thymidylate synthase (TYMS) 2.1.1.45 ST-0236 Irreversible THF (folate)
    In dUMP

    Out dTMP
    › Notes

    Thymidylate synthase (TYMS) catalyzes dUMP + 5,10-methylene-THF → dTMP + dihydrofolate (DHF). 5,10-Methylene-THF donates both the one-carbon unit and the reducing equivalents for conversion of C5 of uracil to the methyl group of thymine. This reaction is effectively irreversible and is the committed step in de novo thymidylate synthesis.

14. 14

    DHF → THF; THF → 5,10-methylene-THF

    ↪ THF; THF → 5,10-methylene-THF

    Dihydrofolate reductase (DHFR) 1.5.1.3 ST-0237 Irreversible/directional NADP+ NADPH THF (folate)
    In Dihydrofolate (DHF)

    Out Tetrahydrofolate (THF)
    › Notes

    Dihydrofolate reductase (DHFR) regenerates THF: DHF + NADPH + H+ → THF + NADP+. Serine hydroxymethyltransferase then catalyzes THF + serine ⇌ 5,10-methylene-THF + glycine, using pyridoxal phosphate; this restores the one-carbon donor required by thymidylate synthase.

Showing all 14 steps.

Overview

Pyrimidine de novo synthesis first constructs the six-membered pyrimidine ring and then attaches it to PRPP, in contrast to purine synthesis, which builds the ring directly on ribose. The pathway supplies UMP, the precursor of UTP, CTP, and—after ribonucleotide reduction and folate-dependent methylation—dTMP. It is essential in proliferating tissues, for RNA synthesis, for DNA precursor production, and for synthesis of UDP-sugars and activated lipid intermediates.

Cellular location

Most reactions are cytosolic and are organized in mammalian cells by the multifunctional CAD polypeptide (carbamoyl-phosphate synthetase II, aspartate transcarbamylase, and dihydroorotase). Dihydroorotate dehydrogenase (DHODH) is anchored to the outer surface of the inner mitochondrial membrane, with its catalytic domain facing the intermembrane space and using the ubiquinone pool as electron acceptor. UMP synthase, nucleotide kinases, CTP synthetase, and thymidylate synthase are cytosolic/nuclear; flux is high in liver, intestinal mucosa, bone marrow, activated lymphocytes, fetal tissues, and neoplasms.

Net energetics

Synthesis of UMP from glutamine, bicarbonate, aspartate, and ribose 5-phosphate requires two ATP → ADP reactions in CPS-II plus one ATP → AMP equivalent to form PRPP: four high-energy phosphate equivalents. Phosphorylation of UMP to UTP consumes two further ATP → ADP equivalents, so UTP costs six high-energy phosphate equivalents from ribose 5-phosphate; conversion of UTP to CTP consumes one more ATP → ADP and one glutamine, yielding a cost of seven equivalents for CTP. Formation of dTMP requires reduction of a ribonucleotide precursor (normally one NADPH-equivalent through the RNR system), one 5,10-methylene-THF molecule converted to DHF, and one NADPH to regenerate THF through DHFR; the detailed cost depends on the dUMP-producing route.

Clinical significance

The pathway supplies pyrimidines for nucleic acids and activated UDP/CTP derivatives used in glycogen, glycoprotein, phospholipid, and sialic acid metabolism. Hereditary UMP synthase deficiency causes orotic aciduria with megaloblastic anemia, growth/developmental impairment, and marked urinary orotate; it is treated with uridine, which bypasses the block and replenishes UMP. DHODH inhibition by teriflunomide suppresses activated lymphocyte proliferation and is used in multiple sclerosis. 5-fluorouracil is converted to 5-fluoro-dUMP, which inhibits thymidylate synthase, while antifolates inhibit DHFR and thereby deplete reduced folate needed for dTMP synthesis.