Nucleic acid metabolism

Pyrimidine and purine metabolism
(KEGG pathway overview MAP01140

Nucleic acids are important intracellular signaling molecules and coenzymes, are the single most important means of coupling endergonic to exergonic reactions, and are the storage of genetic information in the form of DNA and RNA. The latter is composed of nucleotides containing a phosphoribosyl component and either one of the aromatic base adenine (A), guanine (G), cytosine (C), and uracyl (U). These bases can be distinguished by their nitrogen containing, aromatic ring structures and come in two forms - pyrimidines and purines .

Nucleotides are derived from biosynthetic precursors of carbohydrate and amino acid metabolism, and from ammonia and carbon dioxide. The liver is the major organ of de novo synthesis of  all four nucleotides. Degradation in humans, however, is only complete for pyrimidines (C, T, U), but not purines (G, A), which are excreted from the body in form of uric acid (C00366). De novo synthesis of pyrimidines and purines follows two different pathways. Pyrimidines are synthesized first from aspartate and carbamoyl-phosphate in the cytoplasm to the common precursor ring structure orotic acid (C00295), onto which a phosphorylated ribosyl unit is covalently linked. Purines, however, are first synthesized from the sugar template onto which the ring synthesis occurs.

Pyrimidine synthesis
(KEGG pathway MAP00240)

The synthesis of the pyrimidines CTP and UTP occurs in the cytoplasm and starts from the condensation of aspartate with carbamoyl-phosphate to form orotic acid. The enzyme Aspartate carbamoyltransferase (EC forms N-carbamoyl-aspartate (C00438) which is converted into dihydroorotic acid (C00337) by Dihydroorotase (EC The latter is converted to orotate (C00295) by Dihydroorotate oxidase (EC The net reaction is:

                    (S)-Dihydroorotate + O2 = Orotate + H2O2

The formation of cytidine triphosphate in E.coli is controlled by feedback (end product) inhibition.  As a critical concentration of CTP is building up, CTP slows down its own formation by inhibiting Aspartate carbamoyltransferase. This enzyme is composed of two large subunits, one of which carries the catalytic site and the other the regulatory site.

Orotic acid is covalently linked with a phosphorylated ribosyl unit. The activated precursor is PRPP (C00119; 5-Phosphoribosyl 1-pyrophosphate). The covalent linkage between the ribose and pyrimidine occurs at position C1 of the ribose unit, which contains a pyrophosphate,  and N1 of the pyrimidine ring.  Orotate phosphoribosyltransferase (EC catalyzes the net  reaction:

Orotate + 5-Phospho-alpha-D-ribose 1-diphosphate  =  Orotidine 5'-phosphate + Pyrophosphate

Orotidine-5-phosphate (C01103) is decarboxylated by Orotidine-5'-phosphate decarboxylase (EC The enzyme from higher eucaryotes is identical with EC, meaning that the PRPP transferase catalyzes both the ribosylation and decarboxylation reactions, forming UMP from orotic acid in the presence of PRPP. The monophosphonucleotide is phosphorylated by two kinases. First the diphosphate form UDP is produced, which in turn is phosphorylated to UTP. Both steps are fueled by ATP hydrolysis:

                                         ATP + UMP = ADP + UDP
                                         UDP + ATP = UTP + ADP

CTP is subsequently formed by amination of UTP by the catalytic activity of CTP synthetase (EC Glutamine is the NH3 donor and the reaction is fueled by ATP hydrolysis, too:

                    UTP + Glutamine + ATP + H2O = CTP + ADP + Pi

Purine synthesis
(KEGG pathway MAP00230)

Purine ring synthesis starts from the activated ribose PRPP with the sequential addition of nitrogen and carbon containing units, donated from either glutamine (N), glycine (N&C), aspartate (N), folic acid (C1), or CO2.

1. This is the committed step in purine synthesis: glutamine donates an NH2 group displacing the pyrophosphate at ribose C1.
2. Next, a glycine is incorporated fueled by ATP hydrolysis. The carboxyl group forms an amine bond to the NH2 introduced in step 1.
3. A one-carbon unit from folic acid coenzyme N10-formyl-THF is added to the amino group of the substituted glycine followed by the closure of the imidazole ring.
4. Next, a second NH2 group is transferred from a glutamine to the first carbon of the glycine unit.
5. A carboxylation of the second carbon of the glycin unit is concomittantly added.
6. This new carbon is modified by the additional of a third NH2 unit, this time transferred from an aspartate residue.
7. Finally, a second one-carbon unit from formyl-THF is added to the nitrogen group and the ring covalently closed to form the common purine precursor inosine monophosphate (IMP; C00130).

Because of the addition of one-carbon units, nucleic acid synthesis is strictly dependent on folic acid (vitamin). Folic acid is the precursor for the activated one-carbon unit donor tetrahydrofolate (THF). The major source of carbon units for nucleic acid synthesis comes from serine. In this reaction, serine is converted to glycine, while one methylene group is covalently linked to THF to form 5,10-methylene-tetrahydrofolate. This reaction is catalyzed by a mitochondrial enzyme. For more details see one carbon pool metabolism.

Inosine monophosphate is converted to adenosine monophosphate in two steps. First, GTP hydrolysis fuels the addition of aspartate to IMP by adenylosuccinate synthase (EC, substituting the carbonyl oxygen for a nitrogen and forming the intermediate adenylosuccinate. Fumarate is cleaved off forming adenosine monophosphate. This step is catalyzed by adenylosuccinate lyase (EC

GMP is formed by oxidation of IMP forming xanthylate, followed by the insertion of an amino group at C2.  NAD+ is the electron acceptor in the oxidation reaction. The amide group transfer from glutamine is fueled by ATP hydrolysis.

Purine synthesis is regulated by feed back inhibition by the end products. Analogous to amino acid synthesis, the IMP branching point is regulated. The branch leading to AMP is inhibited by AMP, the branch leading to GMP is inhibited by GMP. Note that the catalytic steps are fueled by nucleotide hydrolysis in a reciprocal fashion. GTP is used for AMP formation and ATP is used for GMP formation. This way, the cell has a control over the relative levels synthesize for each nucleotide.

Uric acid formation

While pyrimidine rings can be degraded completely to CO2 and NH3 (urea excretion), purine rings are degraded to the metabolically inert uric acid (C00366). GMP is split into the base guanine and ribose. Guanine is deaminated to xanthine which in turn is oxidized to uric acid. This last reaction is irreversible. Similarly, AMP is deaminated to IMP from which the ribose unit is removed to form hypoxanthine. Hypoxanthine is oxidized to xanthine and finally to uric acid. Instead of uric acid secretion, guanine and IMP can be used for recycling purposes and nucleic acid synthesis in the presence of PRPP and aspartate (NH3 donor).

If the proper disposal of uric acid is impaired, it can cause inflammations, joint pain (arthritis), and kidney problems. This is a condition called gout and is enhanced by low pH conditions, where uric acid is less soluble and starts to crystallize. Gout can be cause by enzymatic defects in guanine recycling and the accumulation of xanthine, which leads to excess uric acid synthesis.

Formation of deoxyribonucleotides for DNA synthesis

Deoxyribonucleotides are synthesized from their ribonucleotide-diphosphates (DNP) by reduction of their C2 ribose carbon. Enzymes catalyzing this reaction are called ribonucleotide reductases (diphosphate reductase EC The 2'-OH hydroxyl-group of ribose is replaced by a hydrogen atom (H) via a free radical reaction.

The reaction steps of the E.coli class I enzyme can be summarized as follows:

1. The active site of the enzyme contains two tyrosine radicals that are stabilized by binuclear Fe-O2-Fe complexes. The tyrosine free radicals abstract an H atom from the C3' position of the diphosphate nucleotide substrate (DNP), forming a free radical substrate intermediate.
2. Acid catalyzed cleavage of the C2'-OH bond involving a sulfhydryl acid-base reaction releases an H2O molecule and a radical-cation intermediate (DNP+).
3. The radical-cation intermediate is reduced by a second cystein pair to yield 3'-deoxynucleotide radical intermediate.
4. This 3' radical abstracts the hydrogen from the tyrosine restoring the initial free radical tyrosine active site and the reduced 2'-deoxyribonucleotide (dDNP).
5. The reductase is now in an oxidized state with a cysteine pair, which has to be reduced to regenerate the initial free cysteine sulfhydryls. This regeneration of the reduced form of ribonucleotide reductase is controlled by thioredoxin, a monomeric, small protein (108 amino acids), which contains a pair of closely spaced cysteine residues. Reduced thioredoxin reduces oxidized reductase (it donates reducing equivalents to the cystein pairs on the reductase). Thioredoxin is regenerated by thioredoxin reductase (EC, which transfers electrons from its FADH2 coenzyme to thioredoxin. The primary electron donor in this reaction is NADPH.

Regulation of ribonucleotide reduction is controlled by allosteric feedback mechanisms. The levels of dATP inhibits binding of all NDPs to the reductase. In addition, there is a multiple cross over inhibition and activation of all dNTP molecules promoting the even synthesis of DNA precursors. The absence of any of these dNTPs is lethal for cells.

Synthesis of dTMP

DNA contains a nucleotide base component not found in RNA. This is thymine and is derived from methylation of dUMP to dTMP (C00364). The deoxyuracylmonophosphate is first formed by dephosphorylation from dUTP by dUTP diphosphorylase (EC dTMP is then synthesized from dUMP by thymidylated synthase (TS; EC with N5,N10-methylene-THF as the methyl donor.

During this methylene transfer, the CH2 is reduced to a methyl group at the expense of the oxidation of tetrahydrofolate (THF) to dihydrofolate (DHF). This mechanism is distinctly different from the 'simpler' methyl group transfer reaction using N5-methyl-THF. DHF needs to be reduced in order to be recycled with an activated one carbon unit. The levels of the amino acid serine and glycine are linked to nucleic acid synthesis showing again the close inter-relationship between the metabolism of the four major macromolecular systems in living organisms.

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