Biological Chemistry - Berezov, T. T., Korovkin, B. F. 1998

Metabolism of Complex Proteins
Nucleic Acid Metabolism
Biosynthesis of Pyrimidine Nucleotides

The Mechanism of pyrimidine nucleotide synthesis was almost fully elucidated through the research of P. Reichard. It has been demonstrated that in animal Cells and microorganisms, the final products of synthesis are likewise not free pyrimidine bases; instead, the ribose moiety attaches to the already formed pyrimidine ring. Synthesis begins from basic precursors (CO₂, NH₃, aspartate), with orotic acid playing a specific and pivotal role.

The sequence of Chemical Reactions involved in the synthesis of pyrimidine NUCLEOTIDES, specifically UMP, can be represented as follows:

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As can be seen, The First stage of UMP synthesis involves The formation of carbamoyl phosphate from glutamine, a reaction catalyzed by cytoplasmic carbamoyl phosphate synthetase (see Chapter 12).

In the second stage, carbamoyl phosphate reacts with aspartate to yield N-carbamoylaspartic acid. The latter undergoes cyclization (mediated by dihydroorotase) with the elimination of a Water molecule, forming dihydroorotic acid, which is subsequently dehydrogenated to yield orotic acid. This reaction requires a specific NAD-dependent enzyme, dihydroorotate dehydrogenase. Orotic acid reversibly reacts with PRPP, acting as a ribose-phosphate donor, to form orotidine-5'-phosphate (OMP). Decarboxylation of OMP leads to the Formation of the first pyrimidine nucleotide—uridine-5'-phosphate (UMP).

The conversion of UMP into UDP and UTP proceeds via phosphotransferase reactions, similarly to purine nucleotides:

УМФ + АТФ <=> УДФ + АДФ;

УДФ + АТФ <=>УТФ + АДФ.

Biosynthesis of cytidine nucleotides. The direct precursor of cytidine nucleotides is UTP, which is converted into CTP:

In prokaryotes, this reaction predominantly utilizes free ammonia, whereas in animal cells, CTP synthetase catalyzes the incorporation of the amide group of glutamine into the 4th position of the UTP pyrimidine ring. It is noteworthy that the resulting CTP acts as a negative effector of the regulatory allosteric enzyme aspartate carbamoyltransferase, inhibiting the initial stage of Pyrimidine Nucleotide Biosynthesis via feedback inhibition. ATP prevents this inhibitory effect.

Biosynthesis of thymidylate nucleotides. Thymidylate nucleotides are constituents of DNA, which contains deoxyribose. Therefore, we must first examine the mechanisms of deoxyribonucleotide synthesis. Using the radiolabeled tracer method, it was shown that this synthesis does not start from free deoxyribose, but rather through the direct reduction of ribonucleotides at the 2'-carbon atom. Upon incubation of labeled precursors (ribonucelotides) in a Cell-free bacterial system, the label was recovered within deoxyribonucleotides. According to P. Reichard's findings, in E. coli all four ribonucleoside diphosphates are reduced to their respective deoxy analogs—dADP, dGDP, dCDP, and dUDP—with the participation of a complex enzyme system comprising at least four distinct Enzymes.

The chemical essence of converting ribonucleotides into deoxyribonucleotides boils down to an elementary event: the reduction of ribose to 2-deoxyribose, which requires two hydrogen atoms. The direct source of these hydrogen atoms is a reduced, thermostable protein called thioredoxin, which contains two free SH groups per 108 amino acid residues. Thioredoxin is readily oxidized, converting into the disulfide S-S form. For its reduction, the system utilizes a specific FAD-containing enzyme, thioredoxin reductase (molecular mass 68,000), which requires NADPH. Denoting ribonucleoside diphosphate conventionally as RDP, the Formation of Deoxyribonucleotides can be outlined as follows:

Both stages can be represented as a scheme:

The synthesis of thymidylate nucleotides requires, In addition to deoxyribose, a methylated derivative of uracil: thymine. It has been established that cells contain a specific enzyme, thymidylate synthase, which catalyzes the methylation not of free uracil, but of dUMP; the reaction proceeds According to the equation:

The methyl group donor in the thymidylate synthase reaction is N⁵,N¹⁰-methylene-THF, which simultaneously provides a hydrogen proton; consequently, one of the final products is dihydrofolic acid (DHF) rather than tetrahydrofolic acid. DHF is subsequently reduced back to THF through the action of an NADPH-dependent Dihydrofolate Reductase. From the resulting TMP, phosphotransferase reactions yield dTDP and dTTP.

The regeneration of N⁵,N¹⁰—CH₂—THF, or rather its biosynthesis, is of particular interest. It has been shown that this synthesis requires The amino acid Serine (serving as the methyl group donor) and a Pyridoxal phosphate-dependent enzyme, serine hydroxymethyltransferase, in accordance with the equation:

The synthesis of all other deoxyribonucleoside 5'-triphosphates directly involved in METABOLISM/36.html">DNA Replication is likewise accomplished by the phosphorylation of deoxyribonucleoside 5'-diphosphates in the presence of ATP:

Next, two diagrams summarize data on The interconversions of purine and pyrimidine nucleotides, as well as their connection to nucleic acid synthesis. As seen from the diagrams, PRPP plays a specific role in the formation of both purine and pyrimidine nucleotides, acting as the phosphoribosyl group donor in The biosynthesis of both orotidine-5'-phosphate and IMP; the latter are considered key substrates in cellular nucleic acid synthesis.