Human Biochemistry, Volume 2 - Murray R. 1993

Structure, Function, and Replication of Information Macromolecules
Metabolism of Purine and Pyrimidine Nucleotides
Purines

Biosynthesis of Purine NUCLEOTIDES

In humans and other mammals, Purine nucleotides are synthesized to meet the body's demand for monomeric precursors of Nucleic Acids, as well as for compounds performing other Functions described in Chapter 34. In some vertebrates (birds, amphibians, reptiles), the Synthesis of purine nucleotides serves an additional function: it is part of the mechanism for excreting excess nitrogen in the form of uric acid; such organisms are termed uricotelic. Organisms in which the end product of Nitrogen METABOLISM is urea (such as humans) are called ureotelic. Because uricotelic organisms eliminate "excess" nitrogen as uric acid, the synthesis of purine nucleotides proceeds more intensively in them than in ureotelic ones. At the same time, the de novo pathways of purine nucleotide synthesis are common to both groups of organisms.

Information regarding THE ORIGIN OF each atom in the purine base molecule was obtained through radioisotope studies conducted on birds, rats, and humans (Fig. 35.2). Figure 35.3 illustrates The pathway of purine nucleotide biosynthesis. The First stage (reaction 1) is The formation of 5-phosphoribosyl-1-pyrophosphate (PRPP). This reaction is not unique to purine nucleotide biosynthesis. PRPP also serves as a precursor in the synthesis of pyrimidine nucleotides (see Fig. 35.15) and is required for the synthesis of NAD and NADP, two Coenzymes containing nicotinic acid.

In reaction 2 (Fig. 35.3), catalyzed by phosphoribosyl pyrophosphate amidotransferase, PRPP and glutamine react to form glutamate and 5-phosphoribosylamine. Although alternative mechanisms for 5-phosphoribosylamine synthesis are possible, the reaction catalyzed by amidotransferase is of the greatest physiological significance in mammalian Tissues.

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Fig. 35.2. Water/144.html">Origin of the nitrogen and carbon atoms of the purine ring.

Next, 5-phosphoribosylamine reacts with Glycine (reaction 3) to yield glycinamide ribosylphosphate (glycinamide ribonucleotide, GAR). The amide group of glutamine provides the nitrogen atom at position 9 of the purine molecule (N-9), while glycine serves as the source of carbon atoms at positions 4 and 5 (C-4 and C-5) of the purine ring. This reaction is catalyzed by glycinamide synthetase. In reaction 4, the N₇ nitrogen atom of glycinamide ribosylphosphate is formylated by N⁵, N¹⁰-methenyltetrahydrofolate. As a result of this reaction, catalyzed by glycinamide ribosylphosphate formyltransferase, the incoming single-carbon unit occupies the C-8 position in the forming purine base. Reaction 5 once again involves glutamine as the donor of the amide group. Amidation occurs at the C-4 atom of formylglycinamide ribosylphosphate and is catalyzed by formylglycinamidine ribosylphosphate synthetase. The attached nitrogen atom will occupy position 3 in the purine molecule.

Closure of the imidazole ring, catalyzed by aminoimidazole ribosylphosphate synthetase, yields aminoimidazole ribosylphosphate (reaction 6). The synthesis then proceeds through the formation of aminoimidazole carboxylate ribosylphosphate (reaction 7). This reaction generates a carbonyl group, the source of which is the CO₂ molecule produced during Respiration.

The nitrogen atom at position 1 originates from the $\alpha$-amino group of aspartate (reaction 8), with the remainder of the molecule forming the succinyl moiety in aminoimidazole succinylcarboxamide ribosylphosphate (SAICAR).

In reaction 9, the succinyl group of SAICAR is removed as fumarate. The remaining aminoimidazole carboxamide ribosylphosphate is formylated (reaction 10) by N¹⁰-formyltetrahydrofolate (f₁₀-H₄folate) to form aminoimidazole carboxamide ribosylphosphate; the reaction is catalyzed by the corresponding formyltransferase. The newly attached carbon atom, like the C-8 atom, is derived from the one-carbon pool with the participation of tetrahydrofolate and occupies position 2 in the purine molecule.

Ring closure (reaction 11) is mediated by IMP cyclohydrolase, yielding the first purine nucleotide, inosinic acid (inosine monophosphate; IMP).

Fig. 35.3. De novo biosynthesis pathway of purines from ribose-5-phosphate and ATP (see text for details). Р — Р0^2-₃ or РО^-₂.

Significance of Folate Metabolism

During The biosynthesis of purine nucleotides (Fig. 35.3), the carbon atoms at positions 8 and 2 are contributed by N⁵, N¹⁰-methenyltetrahydrofolate and N¹⁰-formyltetrahydrofolate, respectively. The latter is formed from N⁵, N¹⁰-methenyltetrahydrofolate, which in turn is the product of NADP-dependent dehydrogenation of N⁵, N¹⁰-methylenetetrahydrofolate. Whereas N⁵, N¹⁰-methylenetetrahydrofolate serves as a source of one-carbon units for numerous acceptors, N⁵, N¹⁰-methenyltetrahydrofolate supplies a one-carbon group (either directly or via the formation of N¹⁰-formyltetrahydrofolate) exclusively for purines. It follows from these data that the inhibition of the formation of these folates exerts a suppressive effect on de novo purine synthesis as well.

Formation of AMP and GMP from IMP

As shown in Fig. 35.4, adenine (reactions 12 and 13) and guanine nucleotides (reactions 14 and 15) are formed via amination, followed by oxidation and amination, of a common precursor—inosine monophosphate (IMP). The amination of IMP proceeds via an intermediate in which aspartate attaches to inosinic acid to form adenylosuccinate. This reaction resembles reaction 8 of Purine Biosynthesis (Fig. 35.3), in which the $\alpha$-nitrogen of aspartic acid provides the N-1 atom of the purine ring. The formation of adenylosuccinate is catalyzed by adenylosuccinate synthetase and requires GTP. Removal of the remaining portion of aspartic acid as fumarate yields adenylic acid (adenosine monophosphate; AMP). The Cleavage of fumarate from adenylosuccinate is catalyzed by the enzyme adenylosuccinase. This same enzyme catalyzes the removal of fumarate from aminoimidazole succinylcarboxamide ribosylphosphate (reaction 9).

Guanosine monophosphate (GMP) is likewise formed from IMP in two steps. In the first reaction of this pathway (reaction 14), IMP undergoes oxidation in the presence of NAD and H₂O to yield xanthine monophosphate (XMP). Subsequently, XMP is aminated by the amide group of glutamine (reaction 15). This process requires ATP, somewhat mirroring the GTP requirement in The conversion of IMP to AMP.

Fig. 35.4. Conversion of IMP to AMP and GMP (see text for details).

Inhibitors of Purine Biosynthesis

Several antimetabolites—glutamine analogues—exert a potent inhibitory effect on purine biosynthesis. Azaserine (O-diazoacetyl-L-Serine) acts as a glutamine antagonist, particularly in reaction 5. Diazonorleucine ([6-diazo-5-oxo]-L-norleucine) blocks reaction 2, while 6-mercaptopurine, alongside other effects, inhibits reactions 13 and 14 of AMP and GMP synthesis, respectively. Mycophenolic acid suppresses reaction 14.

Formation of Purine Nucleoside Di- and Triphosphates

The conversion of AMP and GMP into their respective di- and triphosphates occurs in two stages (Fig. 35.5). Phosphorylation reactions—involving the Transfer of phosphate groups from ATP—are carried out by nucleoside monophosphate kinase and nucleoside diphosphate kinase.

Synthesis of Purine Deoxyribonucleotides

The synthesis of purine and pyrimidine deoxyribonucleotides occurs through the direct reduction of the 2'-carbon of the ribose moiety of the corresponding ribonucleotide, rather than via de novo synthesis from the 2^’-deoxy analogue of PRPP. The reduction of the 2'-carbon atom of ribose takes place only after purine and Pyrimidine nucleotides are converted into their respective nucleoside diphosphates. In some Bacteria, cobalamin (vitamin B₁₂) participates in this reduction process. In animals, the reduction proceeds even in the absence of vitamin B₁₂. The reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates is catalyzed by Ribonucleotide reductase and requires the participation of thioredoxin (a protein cofactor), thioredoxin reductase (a flavoprotein enzyme), and NADPH (a cofactor). The immediate electron donor for the nucleotide is thioredoxin, which is first reduced by NADPH. The reversible redox conversion of thioredoxin is catalyzed by thioredoxin reductase. The reduction of ribonucleoside diphosphate by reduced thioredoxin is catalyzed by ribonucleoside reductase (Fig. 35.6). This complex enzyme system functions in Cells only during periods of active DNA Synthesis AND Cell Division.

Fig. 35.5. Phosphorylation reactions of nucleoside monophosphate and nucleoside diphosphate.

Fig. 35.6. Reduction of ribonucleoside diphosphate to 2'-deoxyribonucleoside diphosphate.

Tissue Specificity of Purine Biosynthesis

Purine nucleotide synthesis de novo does not occur in all human tissues. Erythrocytes and polymorphonuclear leukocytes are incapable of synthesizing 5-phosphoribosylamine, and therefore require exogenous purines for purine nucleotide formation. Peripheral lymphocytes are capable of synthesizing small amounts of purines de novo. It has been established that mammalian Brain cells contain very low amounts of PRPP amidotransferase; based on this, it was concluded that purine nucleotide synthesis in the brain depends on the uptake of exogenous purines. It turned out that the Liver is the primary site of purine nucleotide synthesis in the mammalian Organism. From the liver, free bases or nucleosides are transported to other tissues incapable of de novo purine synthesis.

Purine Salvage Pathways

Purine nucleotide salvage is ensured by Two main mechanisms. Quantitatively, the most important mechanism is the phosphoribosylation of free purine bases by Enzymes that utilize PRPP as a phosphoribose donor. The second general mechanism is the phosphorylation of purine nucleosides at the 5'-hydroxyl group.

1. Phosphoribosylation of Purine Bases

In human tissues, two enzymes carry out the phosphoribosylation of purine bases. The first, adenine phosphoribosyltransferase, transfers phosphoribose from PRPP to adenine, forming AMP (Fig. 35.7). The second, hypoxanthine-guanine phosphoribosyltransferase, catalyzes the phosphoribosylation of xanthine and guanine to yield IMP and GMP, respectively (Fig. 35.8). As will be shown below, the process involving the second enzyme proceeds more actively than the synthesis of AMP from adenine.

Fig. 35.7. Phosphoribosylation of adenine catalyzed by adenine phosphoribosyltransferase.

2. Phosphorylation of Purine Ribonucleosides

The conversion of purine ribonucleosides into purine ribonucleotides in humans is catalyzed by the enzyme adenosine kinase (Fig. 35.9). Additionally, adenosine kinase phosphorylates 2'-deoxyadenosine and also exhibits some activity toward guanosine, inosine, and their 2'-deoxy derivatives. In addition to phosphorylating 2'-deoxycytidine, deoxycytidine kinase catalyzes the phosphorylation of 2'-deoxyadenosine and 2'-deoxyguanosine to form dAMP and dGMP.

Furthermore, human tissues feature a cycle (Fig. 35.10) in which IMP, GMP, and their deoxyribonucleotide analogues are first converted into the corresponding nucleosides (inosine, deoxyinosine, guanosine, and deoxyguanosine) by the action of purine 5'-nucleotidase. Subsequently, through a reaction catalyzed by purine nucleoside phosphorylase, hypoxanthine or guanine is formed along with the phosphorolysis products ribose-1-phosphate or 2'-deoxyribose-1-phosphate. Next, with the participation of PRPP, the cycle is completed by the phosphoribosylation of the resulting bases to IMP or GMP. The physiological function of this cycle remains unknown; however, there is no doubt that PRPP consumption in The Human Body is higher in this cycle than during de novo purine nucleotide synthesis.

Fig. 35.8. Phosphoribosylation of hypoxanthine and guanine to IMP and GMP, respectively. Both reactions are catalyzed by hypoxanthine-guanine phosphoribosyltransferase.

Fig. 35.9. Phosphorylation of adenosine to AMP by adenosine kinase.

A side pathway of this cycle involves the conversion of IMP to AMP (reactions 12 and 13, Fig. 35.4) and the subsequent reaction forming adenosine from AMP. This reaction is apparently catalyzed by the same purine 5'-nucleotidase that hydrolyzes IMP to inosine. The resulting adenosine is then either phosphorylated to AMP by adenosine kinase or converted to inosine by adenosine deaminase. Quantitatively, this "inosine loop" is less significant than the cycle described above, yet the adenosine deamination reaction is crucial for immune system function.

Regulation of Purine Biosynthesis

Fig. 35.10. Purine salvage cycles involving The interconversions of AMP, IMP, and, to a lesser extent, GMP; the Formation of the corresponding ribonucleosides; and their conversion into purine ribonucleotides. Deoxyadenosine, deoxyinosine, and deoxyguanosine are converted via the same pathways; deoxyadenosine and deoxyguanosine can be directly phosphorylated to dAMP and dGMP, respectively.

The synthesis of one IMP molecule requires the Hydrolysis energy of six high-energy phosphodiester ATP bonds, utilizing glycine, glutamine, methenyltetrahydrofolate, and aspartate as precursors. To conserve energy and nutritional resources, efficient Regulation of the de novo purine biosynthesis process is essential. The intracellular concentration of PRPP plays a pivotal role in this process, being determined by the balance between its rates of synthesis, utilization, and degradation. The rate of PRPP synthesis depends on 1) the availability of synthetic substrates, especially ribose-5-phosphate, and 2) the catalytic activity of PRPP synthetase, which in turn correlates with the intracellular concentration of phosphates as well as purine and pyrimidine ribonucleosides acting as Allosteric regulators (Fig. 35.11). The rate of PRPP utilization largely depends on the intensity of the purine salvage cycle, during which xanthine and guanine are phosphoribosylated to the corresponding ribonucleotides. To a lesser extent, the rate of PRPP utilization depends on the intensity of de novo purine synthesis. This Conclusion is supported by the observation that in erythrocytes and cultured fibroblasts of males with a hereditary deficiency in hypoxanthine-guanine phosphoribosyltransferase activity, PRPP levels are elevated several-fold.

Fig. 35.11. Regulation of de novo purine synthesis rate. Solid lines indicate the pathway of chemical transformations. Dashed lines indicate feedback inhibition (⊝) by end products.

Fig. 35.12. Regulation of IMP conversion into adenosine and guanosine nucleotides. Solid lines indicate the pathway of chemical transformations. Dashed lines indicate positive (⊕) and negative (⊝) feedback regulation.

PRPP amidotransferase, the first enzyme involved in the de novo purine nucleotide synthesis pathway, has been shown to be inhibited in vitro by purine nucleotides (particularly adenosine monophosphate and guanosine monophosphate) via a feedback mechanism. These inhibitors compete with the substrate, PRPP, which has been found to play a central role in The regulation of de novo purine synthesis. Much indirect evidence suggests that The Role of amidotransferase in this process is less significant than that of PRPP synthetase.

The formation of GMP or AMP from IMP is regulated by two mechanisms (Fig. 35.12). AMP regulates The activity of adenylosuccinate synthetase, exerting feedback control over its own synthesis. GMP regulates its own synthesis by acting through the same principle on IMP dehydrogenase. Along with this, the formation of adenylosuccinate from IMP on the pathway to AMP is stimulated by GTP. Conversely, the formation of GMP from xanthosine monophosphate requires the presence of ATP. Thus, There is a significant Cross-Regulation of the divergent pathways of IMP metabolism. Such regulation inhibits the biosynthesis of one purine nucleotide when the other is deficient. Hypoxanthine-guanine phosphoribosyltransferase, which catalyzes the formation of IMP and GMP from xanthine and guanine, respectively, is highly sensitive to the inhibitory action of these nucleotides.

The reduction of ribonucleoside diphosphates to deoxyribonucleoside diphosphates is subject to complex regulation. This process (Fig. 35.13) ensures the balanced production of deoxyribonucleotides required for DNA synthesis.

Fig. 35.13. Regulation of the reduction of purine and pyrimidine ribonucleotides to their respective 2'-deoxyribonucleotides. Solid lines indicate the pathway of chemical transformations. Dashed lines indicate positive (⊕) and negative (⊝) feedback regulation.

Purine Catabolism

The end product of purine catabolism in humans is uric acid. Examinations of patients with hereditary deficiencies in the enzyme systems of purine catabolism have established that 99% of uric acid is formed from substrates of nucleoside phosphorylase, which functions in the purine salvage cycle. The purine products of the nucleoside phosphorylase reaction—hypoxanthine and guanine—are converted into uric acid; the intermediate product is xanthine, formed in Reactions Catalyzed by guanase and xanthine oxidase (see Fig. 35.1) in the liver, Small Intestine, and Kidneys.

Xanthine oxidase represents a crucial target for pharmacological intervention in hyperuricemia and Gout. In lower primates and other mammals (but not in humans), uric acid is hydrolyzed by uricase to allantoin (Fig. 35.14), a compound that is highly soluble in water. Birds and terrestrial reptiles lack uricase; as the End products of nitrogen (protein) and purine metabolism, they excrete uric acid and guanine.

These organisms have developed a uricotelic system that allows them to conserve the water associated with uric acid when excreting the latter as a precipitate. If urea were their end product of nitrogen metabolism, conserving Hydration water would be impossible, since the solubility of urea in water reaches 10 mol/L (a concentration far higher than can be achieved by renal concentration of urea).

Uric Acid Metabolism in Humans (Gout)

Human uric acid metabolism has been studied using isotopically labeled uric acid and its precursors, glycine and formate. [¹⁵N]-Uric acid was injected intravenously into healthy individuals and patients with gout, a condition characterized by the accumulation of significant amounts of uric acid and its sodium salt in the body. The total amount of uric acid residing in the body's aqueous phase was calculated from the dilution of the injected isotope. This parameter was termed the "miscible urate pool." The mean value of this indicator for 25 examined healthy adult males was 1200 mg (range 866–1578 mg), while in three healthy females it ranged from 541 to 687 mg. In gout patients, the miscible urate pool was significantly higher, varying from 2000 to 4000 mg for patients without tophi (i.e., without sodium urate deposits in soft tissues). In severe gout accompanied by tophi formation, the miscible urate pool reached 31,000 mg. Its turnover rate in healthy individuals is 600 mg per 24 h. About 18–20% of the uric acid removed from the body is degraded to CO₂ and ammonia and excreted through the intestine. A certain amount of urates is excreted in the Bile and undergoes degradation by intestinal microflora. It should be noted that The breakdown of uric acid to CO₂ and NH₃ in humans is not mediated by intestinal bacteria.

Fig. 35.14. Formation of allantoin from uric acid.

The Biological Significance of urates in humans is not limited to their role as the end product of purine metabolism. Urates can function as antioxidants, undergoing non-enzymatic conversion to allantoin. It is hypothesized that the endogenous antioxidant urate replaces ascorbate in primates, as these mammals have lost The ability to synthesize it. Thus, it is quite possible that during evolution, the loss of uricase provided certain selective advantages for organisms that lost the ability to reduce gulonolactone to ascorbate.

Sodium urate is readily filtered by the mammalian renal glomeruli, undergoes extensive reabsorption and partial secretion in the proximal tubules, is then secreted in the Loop of Henle, and is likely reabsorbed again in the distal tubules. A healthy human excretes 400–600 mg of uric acid per day. A large number of pharmacological agents and natural compounds affect the renal tubular reabsorption of sodium urate and its excretion. Aspirin in high doses inhibits both the renal excretion and reabsorption of uric acid.