Metabolism of pyrimidine nucleotides, regulation, inhibitors, disorders

Synthesis

composition

pyrimidine synthesis

In humans, the key step in the synthesis of pyrimidines is the synthesis of carbamoyl phosphate from glutamine and CO2, which is catalyzed by carmoyl phosphate synthase II (CPS II).

Unlike the synthesis of the purine ring, which is synthesized on a pre-existing ribose-5-phosphate, the pyrimidine ring is synthesized before it is attached to the ribose-5-phosphate, which supplies the phosphoribosyl pyrophosphate (PRPP). The source for pyrimidine ring atoms is glutamine, CO2 and aspartic acid.

• CPS II is inhibited by UTP (an end product of the pathway that can be converted to other pyrimidine nucleotides) and activated by PRPP.

• Carbamoyl phosphate, synthesized by CPS I, is a precursor of urea.

• A defect in ornithine transcarbamylase in the ornithine cycle promotes the synthesis of pyrimidines, due to an increase in the level of carbamoyl phosphate.

The second step in pyrimidine synthesis is the formation of carbamoylaspartate, catalyzed by aspartate transcarbamoylase.

The pyrimidine ring is then closed hydrolytically by the action of dihydrooratase. The resulting dihydroorotate is then oxidized to orotic acid (orotate).

• The enzyme that produces orotate, dihydroorotate dehydratase, is located on the inner mitochondrial membrane. All other enzymes of the pathway are located cytosolic.

The first three enzymatic reactions (CPS II, aspartate transcarbamoylase and dihydrooratase) are actually three different catalytic domains of a single polypeptide chain, called CAD;  the name comes from the first letters of all domains.

• This is an example of a multifunctional or multicatalytic polypeptide that mediates the ordered synthesis of important substances.
• Synthesis of the purine nucleotide IMP also requires multifunctional proteins.

The completed pyrimidine ring is converted to the nucleotide orotidine-5'-monophosphate (OMP) in the second part of pyrimidine nucleotide synthesis. In this case too, the ribose-5-phosphate donor is PRPP.  The enzyme orotate phosphoribosyltransferase produces OMP and releases pyrophosphate, making this reaction biologically irreversible.

OMP, the "parent" pyrimidine mononucleotide is converted to uridine monophosphate (UMP) by orotidylate decarboxylase, which removes the acidic carboxyl group.

• Orotate phosphoribosyltransferase and orotidylate decarboxylase are also catalytic domains of one polypeptide chain called UMP-synthase.

UMP is successively phosphorylated to UDP and then UTP.

UDP is a substrate for ribonucleotide reductase, which produces dUDP.  dUDP is phosphorylated to dUTP, which is immediately hydrolyzed to dUMP by UTP-diphosphatase (dUTP-ase).

• dUTP-ase thus plays an important role in the reduction of dUTP as a substrate for DNA synthesis, thereby preventing misincorporation of uracil into DNA.

CTP is produced by amination of UTP using CTP-synthetase and glutamine as a nitrogen source. Some CTPs are dephosphorylated to CDP, which is a substrate for ribonucleotide reductase. The resulting dCDP can be phosphorylated to dCTP for DNA synthesis.

dUMP is converted to dTMP by thymidylate synthase, which uses N5,N10methylenetetrahydrofolate as a methyl group source. This is an unusual reaction in which tetrahydrofolate (THF) contributes not only one carbon, but also two hydrogen atoms from the pteridine ring, thereby oxidizing THF to dihydrofolate (DHF).

• Thymidylate synthase inhibitors are for example thymine analogues such as  5-fluorouracil, which act as effective cytostatics. 5-flurouracil is metabolically converted to 5-FdUMP, which remains permanently bound to inactivated thymidylate synthase;  for this reason, this drug is also called a "suicide" inhibitor.

DHF can be reduced to THF by dihydrofolate reductase, an enzyme that is inhibited by drugs such as Methotrexate. By reducing the supply of THF, this folate analog not only inhibits purine synthesis, but by preventing the methylation of dUMP to dTMP, it also reduces the concentration of this essential component of DNA synthesis in the cell. If DNA synthesis is inhibited, cell growth also slows down. All the mentioned drugs are therefore used as cytostatics in the treatment of cancer.

pyrimidine salvage pathway

Trimethoprim, a folate analogue, has antibacterial potential as it selectively inhibits bacterial dihydrofolate reductase.

Salvage pathway

A pair of pyrimidine bases is spared (salvage pathway) in human cells. However, pyrimidine nucleotides can be spared by nucleoside kinase, which uses ATP to phosphorylate nucleosides into nucleotides. The sparing of pyrimidine nucleotides is the basis for the use of uridine in the treatment of hereditary orotic aciduria.

Unlike the purine ring, which is not cleaved in human cells, the pyrimidine ring opens and degrades to the very soluble products, β-alanine and β-aminoisobutyrate, producing NH3 and CO2.(PRPP) cytosolic proteins.

Pathobiochemistry

Orotic aciduria is an autosomal recessive impairment of pyrimidine synthesis, caused by impairment of UMP-synthase.

It is characterized by the accumulation of orotate in body fluids and its increased excretion, while crystallization may also occur. At the same time, there is a deficiency of pyrimidines for DNA synthesis resulting in cell division disorders and megaloblastic anemia unresponsive to treatment with iron, vitamin B12 or folic acid, because tissue disorder in insufficient DNA synthesis due to lack of pyrimidine bases.

It is manifested by growth failure, psychomotor retardation, leukopenia and malaise. It is treated with uridine. There are also several defects in pyrimidine ring degradation.

Sources

MATOUŠ, Bohuslav, et al. Základy lékařské chemie a biochemie. 1. vydání. Praha: Galén, 2010. 540 s.