BIOSYNTHESIS OF PURINE RIBONUCLEOTIDES
Many compounds contribute to the purine ring of the nucleotides1. N1 of purine is derived from amino group of aspartate.
2. C2 and C8 arise from formate of N10- formyl THF.
3. N3 and N9 are obtained from amide group of glutamine.
4. C4, C5 and N7 are contributed by glycine.
5. C6 directly comes from CO2.
It should be remembered that purine bases are not synthesized as such, but they are formed as ribonucleotides. The purines are built upon a pre-existing ribose 5-phosphate. Liver is the major site for purine nucleotide synthesis. Erythrocytes, polymorphonuclear leukocytes and brain cannot produce purines.
The pathway for the synthesis of inosine monophosphate (IMP or inosinic acid)
1. Ribose 5-phosphate, produced in the hexose monophosphate shunt of carbohydrate metabolism is the starting material for purine nucleotide synthesis. It reacts with ATP to form phosphoribosyl pyrophosphate (PRPP).2. Glutamine transfers its amide nitrogen to PRPP to replace pyrophosphate and produce 5-phosphoribosylamine. The enzyme PRPP glutamyl amidotransferase is controlled by feedback inhibition of nucleotides (IMP, AMP and GMP). This reaction is the ‘committed step’ in purine nucleotide biosynthesis.
3. Phosphoribosylamine reacts with glycine in the presence of ATP to form glycinamide ribosyl 5-phosphate or glycinamide ribotide (GAR).
4. N10-Formyl tetrahydrofolate donates the formyl group and the product formed is formyl- glycinamide ribosyl 5-phosphate.
5. Glutamine transfers the second amido amino group to produce formylglycinamidine ribosyl 5-phosphate.
6. The imidazole ring of the purine is closed in an ATP dependent reaction to yield 5-amino- imidazole ribosyl 5-phosphate.
7. Incorporation of CO2 (carboxylation) occurs to yield aminoimidazole carboxylate ribosyl 5-phosphate. This reaction does not require the vitamin biotin and/or ATP which is the case with most of the carboxylation reactions.
8. Aspartate condenses with the product in reaction 7 to form aminoimidazole 4-succinyl carboxamide ribosyl 5-phosphate.
9. Adenosuccinate lyase cleaves off fumarate and only the amino group of aspartate is retained to yield aminoimidazole 4-carboxamide ribosyl 5-phosphate.
10. N10-Formyl tetrahydrofolate donates a one-carbon moiety to produce formamino- imidazole 4-carboxamide ribosyl 5-phosphate. With this reaction, all the carbon and nitrogen atoms of purine ring are contributed by the respective sources.
11. The final reaction catalysed by cyclo- hydrolase leads to ring closure with an elimination of water molecule. The product obtained is inosine monophosphate (IMP), the parent purine nucleotide from which other purine nucleotides can be synthe- sized.
Inhibitors of purine synthesis
Folic acid (THF) is essential for the synthesis of purine nucleotides (reactions 4 and 10). Sulfonamides are the structural analogs of para- aminobenzoic acid (PABA). These sulfa drugs can be used to inhibit the synthesis of folic acid by microorganisms. This indirectly reduces the synthesis of purines and, therefore, the nucleic acids (DNA and RNA). Sulfonamides have no influence on humans, since folic acid is not synthesized and is supplied through diet.The structural analogs of folic acid (e.g. methotrexate) are widely used to control cancer. They inhibit the synthesis of purine nucleotides (reaction 4 and 10) and, thus, nucleic acids. Both these reactions are concerned with the transfer of one-carbon moiety (formyl group). These inhibitors also affect the proliferation of normally growing cells. This causes many side-effects including anemia, baldness, scaly skin etc.
Synthesis of AMP and GMP from IMP
Inosine monophosphate is the immediate precursor for the formation of AMP and GMP.Aspartate condenses with IMP in the presence of GTP to produce adenylsuccinate which, on cleavage, forms AMP.
For the synthesis of GMP, IMP undergoes NAD+ dependent dehydrogenation to form xanthosine monophosphate (XMP). Glutamine then transfers amide nitrogen to XMP to produce GMP.
6-Mercaptopurine is an inhibitor of the synthesis of AMP and GMP. It acts on the enzyme adenylsuccinase (of AMP pathway) and IMP dehydrogenase (of GMP pathway).
Formation of purine nucleoside diphosphates and triphosphates
The nucleoside monophosphates (AMP and GMP) have to be converted to the corresponding di- and triphosphates to participate in most of the metabolic reactions. This is achieved by the transfer of phosphate group from ATP, catalyzed by nucleoside monophosphate (NMP) kinases and nucleoside diphosphate (NDP) kinases.Salvage pathway for purines
The free purines (adenine, guanine and hypoxanthine) are formed in the normal turnover of nucleic acids (particularly RNA), and also obtained from dietary sources.The purines can be directly converted to the corresponding nucleotides, and this process is known as ‘salvage pathway’.
Adenine phosphoribosyl transferase catalyzes the formation of AMP from adenine. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) converts guanine and hypoxanthine, respectively, to GMP and IMP. Phosphoribosyl pyrophosphate (PRPP) is the donor of ribose 5-phosphate in the salvage pathway. The salvage pathway is particularly important in certain tissues such as erythrocytes and brain where de novo (a new) synthesis of purine nucleotides is not operative.
A defect in the enzyme HGPRT causes Lesch- Nyhan syndrome.
Regulation of purine nucleotide biosynthesis metabolism of nucleotides
The purine nucleotide synthesis is well coordinated to meet the cellular demands. The intracellular concentration of PRPP regulates purine synthesis to a large extent. This, in turn, is dependent on the availability of ribose 5-phosphate and the enzyme PRPP synthetase.PRPP glutamyl amidotransferase is controlled by a feedback mechanism by purine nucleotides. That is, if AMP and GMP are available in adequate amounts to meet the cellular requirements, their synthesis is turned off at the amidotransferase reaction.
Another important stage of regulation is in the conversion of IMP to AMP and GMP. AMP inhibits adenylsuccinate synthetase while GMP inhibits IMP dehydrogenase. Thus, AMP and GMP control their respective synthesis from IMP by a feedback mechanism.
Conversion of ribonucleotides to deoxyribonucleotides
The synthesis of purine and pyrimidine deoxyribonucleotides occurs from ribo- nucleotides by a reduction at the C2 of ribose moiety.this reaction is catalysed by a multisubunit (two B1 and two B2 subunits) enzyme, ribonucleotide reductase.
Supply of reducing equivalents : The enzyme ribonucleotide reductase itself provides the hydrogen atoms needed for reduction from its sulfhydryl groups. The reducing equivalents, in turn, are supplied by thioredoxin, a monomeric protein with two cysteine residues.
NADPH-dependent thioredoxin reductase converts the oxidized thioredoxin to reduced form which can be recycled again and again. Thioredoxin thus serves as a protein cofactor in an enzymatic reaction.
Regulation of deoxyribonucleotide synthesis : Deoxyribonucleotides are mostly required for the synthesis of DNA. The activity of the enzyme ribonucleotide reductase maintains the adequate supply of deoxyribonucleotides.
The drug hydroxyurea inhibits ribonucleotide reductase by destroying free radicals required by this enzyme. Hydroxyurea is used in the treatment of cancers such as chronic myologenous leukemia.
DEGRADATION OF PURINE NUCLEOTIDES
The end product of purine metabolism in humans is uric acid. The sequence of reactions in purine nucleotide degradation is -1. The nucleotide monophosphates (AMP, IMP and GMP) are converted to their respective nucleoside forms (adenosine, inosine and guanosine) by the action of nucleotidase.
2. The amino group, either from AMP or adenosine, can be removed to produce IMP or inosine, respectively.
3. Inosine and guanosine are, respectively, converted to hypoxanthine and guanine (purine bases) by purine nucleoside phosphorylase. Adenosine is not degraded by this enzyme, hence it has to be converted to inosine.
4. Guanine undergoes deamination by guanase to form xanthine.
5. Xanthine oxidase is an important enzyme that converts hypoxanthine to xanthine, and xanthine to uric acid. This enzyme contains FAD, molybdenum and iron, and is exclusively found in liver and small intestine. Xanthine oxidase liberates H2O2 which is harmful to the tissues. Catalase cleaves H2O2 to H2O and O2.
Uric acid (2,6,8-trioxypurine) is the final excretory product of purine metabolism in humans. Uric acid can serve as an important antioxidant by getting itself converted (non- enzymatically) to allantoin. It is believed that the antioxidant role of ascorbic acid in primates is replaced by uric acid, since these animals have lost the ability to synthesize ascorbic acid.
Most animals (other than primates) however, oxidize uric acid by the enzyme uricase to allantoin, where the purine ring is cleaved. Allantoin is then converted to allantoic acid and excreted in some fishes .
Further degradation of allantoic acid may occur to produce urea (in amphibians, most fishes and some molluscs) and, later, to ammonia (in marine invertebrates).