Biochemistry and Molecular Biology - Belyasova N.A. 2002

Metabolism. Energy-requiring processes
Metabolism of nitrogenous compounds
Nucleotide metabolism

Purine and pyrimidine NUCLEOTIDES, the monomers of Nucleic Acids, are among the most complexly organized metabolites. Their de novo synthesis requires A large number of Enzymes and energy input, yet it can be carried out by all organisms except for certain auxotrophic microorganism mutants. Due to the exceptional complexity of nucleotide synthesis, their breakdown (for example, during the degradation of unneeded nucleic acids) typically does not proceed to completion, but rather stops at specific constituent parts. These breakdown products (primarily purine nitrogenous bases) can be reused to form nucleoside monophosphates. This phenomenon is known as resynthesis.

Complete degradation of purine and pyrimidine nucleotides yields phosphoric acid, carbon dioxide, and ammonia, whereas incomplete degradation yields ribose (or ribose-5-phosphate), nitrogenous bases, urea, allantoic acid, allantoin, and uric acid. As a rule, the lowest forms of life (such as Bacteria) are capable of completely degrading nucleotides, whereas organisms at higher evolutionary stages break down nucleotides and their constituent nitrogenous bases into less simple compounds.

Nucleotide Biosynthesis. This process involves Amino Acids, which serve as sources for most of the carbon atoms and all of the nitrogen atoms within the nitrogenous bases. The Synthesis of purine and pyrimidine nucleotides proceeds via distinct pathways.

The de novo Biosynthesis of Purine nucleotides begins with the stepwise assembly of the nitrogenous base ring on a molecule of 5-phosphoribosyl-1-pyrophosphate, which is also involved in The biosynthesis of Histidine and Tryptophan (Fig. 16.10). First, an amino group (N-9), donated by glutamine, is attached to phosphoribosyl pyrophosphate via a Transamination reaction. Next, two carbon atoms (C-4, C-5) and a nitrogen atom (N-7) derived from Glycine, along with a carbon atom (C-8) from formyltetrahydrofolic acid, are incorporated into the five-membered ring. This is followed by the successive addition of the Components of the six-membered ring: a nitrogen atom (N-3) from glutamine, a carbon atom (C-6) from CO₂, a nitrogen atom (N-1) from aspartate, and a carbon atom (C-2) from formyltetrahydrofolic acid. In the final step, the six-membered ring is closed, forming the molecule of inosine-5'-monophosphate (IMP), the primary intermediate of Purine Biosynthesis. This metabolite is subsequently converted into AMP and GMP (Fig. 16.14).

The synthesis of purine nucleoside monophosphates is allosterically regulated: ADP and GDP inhibit The formation of phosphoribosyl pyrophosphate, while AMP and GMP inhibit The activity of the first enzyme catalyzing the Formation of the nitrogenous base ring—amidophosphoribosyldisulfatase (transamination reaction). Thus, a self-regulation mechanism prevents the overproduction of purine nucleotides. In addition, the ratio between the two purine nucleotides derived from inosine monophosphate is regulated via cross-activation (Fig. 16.14).

Purine nucleoside monophosphates are converted into nucleoside diphosphates with the participation of nucleoside monophosphate Kinases:

Class="center">АМР + АТР → 2ADP GMP + ATP → GDP + ADP

The ADP generated in these reactions can be converted into ATP through substrate-level and Oxidative Phosphorylation, as well as Photophosphorylation. Guanosine diphosphate is converted into guanosine triphosphate with the participation of nucleoside diphosphate kinase:

GDP + ATP → GTP + ADP

Ribonucleotides are converted into deoxyribonucleotides at the nucleoside diphosphate level via a reduction reaction. This multi-step reaction is catalyzed by two enzymes: thioredoxin reductase and nucleoside diphosphate reductase (Ribonucleotide reductase). The hydrogen donor for ribose residue reduction is NADPH, which, however, does not transfer hydrogen directly to ribose, but instead reduces a specialized protein—thioredoxin. During this process, the disulfide bridge within the thioredoxin molecule is cleaved, yielding two sulfhydryl groups that participate in the reduction of the ribose carbon atoms (Fig. 16.15). Nucleoside diphosphate reductase catalyzes the elimination of a Water molecule from the ribose residue with the aid of a Tyrosine radical generated in its active center. As a result, the substrate is converted into a radical cation, which is reduced to a deoxyribonucleotide with the participation of thioredoxin, while the tyrosyl radical is regenerated in the enzyme's active center.

Fig. 16.14. Scheme of purine nucleotide biosynthesis and cross-activation in the Regulation of the AMP-to-GMP ratio. The Origin of the carbon and nitrogen atoms is indicated within the hypoxanthine moiety of the inosine monophosphate nitrogenous base.

Ribonucleotide reductase is allosterically activated by ATP and inhibited by dATP (deoxyadenosine triphosphate), allowing for The regulation of the ratio between oxidized and reduced forms of nucleoside diphosphates.

The de novo BIOSYNTHESIS OF PYRIMIDINE nucleotides proceeds as follows: first, the pyrimidine ring is synthesized, and ribose-5-phosphate is subsequently attached to it. The Synthesis of the pyrimidine ring involves carbamoyl phosphate and aspartate. In turn, carbamoyl phosphate is formed in the Cytoplasm from carbon dioxide and glutamine (the amino group donor), a reaction catalyzed by carbamoyl phosphate synthetase, which hydrolyzes glutamine. The Condensation of carbamoyl phosphate and aspartate (catalyzed by aspartate carbamoyltransferase) forms the six-membered ring intermediate, dihydroorotate. Its oxidation to orotate and The addition of phosphoribosyl pyrophosphate lead to the formation of orotidine-5'-monophosphate, the decarboxylation of which yields uridine-5'-monophosphate (UMP), the primary intermediate of pyrimidine biosynthesis. UMP is then further converted into other pyrimidine nucleotides (Fig. 16.16) via a rather complex pathway.

Fig. 16.15. Scheme of ribonucleotide reduction reactions (details in text)

Fig. 16.16. Scheme of the stages of Pyrimidine Nucleotide Biosynthesis: M-TGF — methylenetetrahydrofolic acid; TGF — tetrahydrofolic acid

Pyrimidine nucleoside triphosphates are formed from nucleoside monophosphates with the participation of ATP, as described above for purine nucleotides. Cytidylate is formed from uridylate at the nucleoside triphosphate level (Fig. 16.16) with the participation of cytidine triphosphate synthase. Deoxythymidylate arises from uridylate at the nucleoside monophosphate level via the methylation of deoxyuridine monophosphate. This reaction is catalyzed by thymidylate synthase with the participation of a methyl group donor, methylenetetrahydrofolate. The reduction of ribonucleotides to deoxyribonucleotides proceeds via the mechanism described previously for Purines (Fig. 16.15).

The Regulation of Pyrimidine nucleotide biosynthesis in bacteria was described earlier (Chapter 6) and is achieved through the Allosteric Regulation of the enzyme aspartate carbamoyltransferase. In animals, the key enzyme in pyrimidine nucleotide biosynthesis is carbamoyl phosphate synthetase, which is allosterically activated by ATP and phosphoribosyl pyrophosphate, and inhibited by UTP.

Thus, the de novo synthesis of purine and pyrimidine nucleotides has been examined. However, this is not the only pathway for their formation; to conserve cellular resources, Cells frequently produce nucleotides via so-called resynthesis. This pathway utilizes free purine and pyrimidine nitrogenous bases—generated during The breakdown of nucleic acids—along with phosphoribosyl pyrophosphate. The reactions leading to the formation of nucleoside monophosphates are catalyzed by the corresponding phosphoribosyltransferases. This mode of nucleotide synthesis is particularly characteristic of malignant tumor cells.

Nucleotide degradation. This process typically concludes the breakdown of nucleic acids, which is catalyzed by Nucleases. Two Types of nucleases are distinguished: exonucleases (which attack polynucleotide chains from the ends) and endonucleases (which cleave covalent bonds within the molecule without requiring free 3'- and 5'-ends). The action of nucleases results in the formation of free nucleotides.

Purine and Pyrimidine nucleotides are degraded via different pathways: the purine ring remains intact and is converted into uric acid or its derivatives, which are excreted from the Organism; pyrimidine nitrogenous bases are broken down into small fragments that can either re-enter metabolic pathways or be excreted.

In primates (including humans), birds, and reptiles, the Catabolism of Purine nucleotides is accompanied by the formation of ribose phosphate (which participates in METABOLISM) and uric acid (which is excreted in the urine). In other mammals and Mollusks, uric acid is oxidized to allantoin, which is then excreted. In teleost fish, allantoin is hydrated to allantoate, whereas in amphibians and cartilaginous ganoids, allantoate is further degraded to urea and glyoxylate. Finally, in microbial cells, purine nucleotides can be broken down into the simplest compounds—ammonia, carbon dioxide, glycine, and formic acid. Simplified schemes of purine nucleotide degradation are shown in Fig. 16.17.

Fig. 16.17. Simplified pathways of purine nucleotide degradation

It is important to note that The conversion of hypoxanthine to xanthine and of xanthine to uric acid is catalyzed by the same enzyme, xanthine oxidase. At each of these stages, an oxo group is introduced into the substrate via oxidation by molecular oxygen, yielding hydrogen peroxide as a byproduct (Fig. 16.17), the toxicity of which is neutralized by peroxidase enzymes.

Unlike urea, uric acid—the end product by which purine nitrogenous bases are excreted from The Human Body—is sparingly soluble in water. Consequently, certain Metabolic Disorders (including specific Hereditary diseases) or the excessive consumption of meat products rich in purines can lead to elevated Blood concentrations of uric acid (hyperuricemia). This condition may result in the deposition of uric acid crystals in the joints (Gout) and other Organs.

The breakdown of pyrimidine nucleotides begins with the removal of a phosphoric acid residue to form a nucleoside. Subsequently, thymidine and uridine are degraded into simpler compounds (Fig. 16.18) through a series of reactions common to both nucleosides. Important intermediates in these pathways are uracil and thymine, which can either participate in the resynthesis of pyrimidine nucleotides or be degraded via a shared mechanism: the pyrimidine ring is first reduced and then cleaved hydrolytically.

It should be noted that one of the amino groups of pyrimidine nitrogenous bases is removed via hydrolytic deamination, while the second is eliminated through transamination, where the amino group acceptor is a-ketoglutarate (converted into glutamate) or Pyruvate (converted into Alanine). In addition to acetate and propionate, the final products of pyrimidine nucleotide degradation include CO₂ and NH₃.

Fig. 16.18. Simplified pathways for the degradation of pyrimidine nucleotides