Nucleotide Metabolism

What are the structures and functions of nucleotides?

• Nucleotides participate in four important biochemical processes: (1) energy conversion reactions, (2) signal transduction pathways, (3) coenzyme-dependent reactions, and (4) genetic information storage and transfer.

• Purine bases contain nine atoms in the heterocyclic ring, whereas pyrimidine bases contain six atoms.

• The two common purine bases are adenine and guanine. The three common pyrimidine bases are cytosine, thymine, and uracil. Uracil is found only in RNA, whereas thymine is found only in DNA.

• Ribonucleotides contain hydroxyl groups at both C-3′ and C-2′ of the ribose, whereas deoxyribonucleotides lack the hydroxyl group at C-2′.

• A nucleoside consists of a base and sugar. A nucleotide is a phosphorylated nucleoside; for example, ATP is adenosine-5′-triphosphate, and AMP is adenosine-5′-monophosphate.

• There are two types of salvage pathways: (1) the nucleotide salvage pathway, which salvages nucleoside monophosphates (NMPs) to generate NTPs; and (2) the nucleotide base salvage pathway, which uses phosphoribosyl pyrophosphate (PRPP) and free nucleotide bases to form nucleoside monophosphates.

How are purine nucleotides synthesized?

• Purine bases are synthesized directly on the ribose sugar, whereas pyrimidine bases are first synthesized as a closed ring before attaching to the ribose sugar.

• The four nitrogen atoms in purine bases are derived from aspartate (N-1), glycine (N-7), and two glutamines (N-3 and N-9). The five carbon atoms come from glycine (C-4 and C-5), HCO₃⁻ (C-6), and two molecules of N¹⁰-formyl-tetrahydrofolate (C-2 and C-8).

• The purine nucleotides AMP and GMP are synthesized from the common intermediate inosine-5′-monophosphate (IMP), which contains the purine base hypoxanthine. The biosynthesis of IMP in E. coli can be broken down into two stages, each consisting of five well-characterized biochemical reactions.

• The first stage of purine biosynthesis generates the five-membered imidazole ring on PRPP in a series of five reactions, leading to the formation of 5-aminoimidazole ribonucleotide (AIR). The second ring of the purine molecule is generated in the second stage by five reactions that convert AIR to IMP.

• A purinosome is a large protein complex in eukaryotic cells that contains all of the enzymatic activities needed to synthesize the purine ring. Evidence for purinosomes comes from human cancer cells grown in purine-depleted media that express fluorescent fusion proteins linked to purine biosynthetic enzymes.

• IMP is the precursor to both AMP and GMP, which are generated in parallel biosynthetic pathways. Balanced regulation of metabolic flux through these two parallel pathways is mediated by GTP and ATP, which are required for AMP and GMP synthesis, respectively.

• AMP and GMP are phosphorylated to generate nucleoside triphosphates for RNA and DNA synthesis. The enzyme nucleoside diphosphate kinase can use any nucleoside triphosphate as a phosphate donor and phosphorylates both nucleoside diphosphates and deoxynucleoside diphosphates.

• Flux through the purine biosynthetic pathway is primarily controlled by inhibition of the PRPP synthetase and glutamine-PRPP amidotransferase reactions. The balance of AMP and GMP synthesis is controlled by both feedback inhibition of the individual branches in the pathway and by ATP and GTP cross-talk regulation.

How does purine degradation to uric acid cause gout?

• Excess AMP, GMP, and IMP nucleotides from cellular or dietary nucleic acids are dephosphorylated by the enzyme 5′-nucleotidase to generate adenosine, inosine, and guanosine, respectively, which are then further degraded into uric acid.

• Uric acid is nearly insoluble in water and is excreted as a dry paste by organisms that need to conserve water, such as birds, reptiles, and insects. Primates also excrete small amounts of uric acid, but most of their nitrogen waste products are excreted as urea produced by the urea cycle. Uric acid is further degraded in some animals to other nitrogen-containing waste products.

• Gout is a metabolic disease caused by deregulation of the purine biosynthetic pathway, resulting in urate accumulation. The pKₐ of uric acid is ~5.7, so most uric acid in blood at pH 7.4 is urate anion, or more commonly, sodium urate.

• Gout is caused by the buildup of uric acid crystals in the joints and kidneys. The big toe is a common joint affected by urate crystals because urate is less soluble at lower temperatures, and the big toe, which is furthest from the heart, is colder than other parts of the body.

• Dietary causes of gout include alcohol, which interferes with urate excretion, and the high amounts of purines contained in meats. Gout has been linked to increased levels of PRPP synthetase, defects in feedback inhibition of glutamine-PRPP amidotransferase, and deficiencies in the salvage enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT).

How do defects in purine enzymes cause Lesch–Nyhan syndrome and ADA-SCID?

• Lesch–Nyhan syndrome is a rare recessive genetic disease caused by defects in HGPRT, which leads to a buildup of guanine and hypoxanthine and is characterized by unusual neurologic symptoms. Lesch–Nyhan syndrome follows the inheritance pattern of an X-linked recessive genetic mutation because the HGPRT gene is located on the X chromosome.

• Defects in the enzyme adenosine deaminase (ADA) cause ADA–SCID (SCID: severe combined immunodeficiency disease), leading to adenosine accumulation, which shunts excess adenosine into dATP production. Because dATP is an inhibitor of the enzyme ribonucleotide reductase, rapidly dividing B and T cells in the immune system fail to proliferate.

• Gene therapy has been used to treat ADA–SCID patients, in which a functional copy of the adenosine deaminase gene is inserted into hematopoietic stem cells (HSCs) isolated from the patient. Healthy ADA-producing cells are identified in culture and then expanded in number and injected back into the patient.

How are pyrimidine nucleotides synthesized and degraded?

• The six atoms in the pyrimidine ring are derived from just two precursor biomolecules: aspartate (C-1, C-4, C-5, C-6) and carbamoyl phosphate, which is generated from glutamine (N-3) and HCO₃⁻ (C-2).

• The pyrimidine biosynthetic pathway in E. coli consists of six reactions to generate the pyrimidine nucleotide UMP, which is converted to UTP by sequential phosphorylation reactions and then aminated by CTP synthetase to generate CTP.

• E. coli purine and pyrimidine biosynthetic enzymes are encoded in the bacterial genome as separate polypeptides, whereas in most animals, including humans, several catalytic activities required for purine and pyrimidine biosynthesis are combined into large multifunctional enzymes.

• Three activities encoded by the multi-subunit CAD enzyme in animals are carbamoyl phosphate synthetase II, aspartate transcarbamoylase, and dihydroorotase. The bifunctional UMP synthase enzyme in animals encodes the orotate phosphoribosyl transferase and OMP decarboxylase activities, whereas the dihydroorotate dehydrogenase enzyme is a single polypeptide that functions in the mitochondrial matrix.

• Pyrimidine biosynthesis is regulated by both feedback inhibition and allosteric activation in bacteria and animal cells. Aspartate transcarbamoylase is the key regulated enzyme in the pyrimidine biosynthetic pathway in E. coli cells, being activated by ATP and inhibited by CTP. Flux through the pyrimidine biosynthetic pathway in animal cells is controlled by allosteric regulation of the CAD enzyme, UMP synthase, and CTP synthetase.

• The first reaction in the pyrimidine degradation pathway is catalyzed by the rate-limiting enzyme dihydropyrimidine dehydrogenase, which converts uracil and thymine to dihydrouracil and dihydrothymine, respectively. Dihydropyrimidine dehydrogenase enzyme deficiencies are present in ~5% of the human population, which makes them fairly common.

• Dihydropyrimidine dehydrogenase deficiency is the cause of drug toxicity in cancer patients being treated with 5-fluorouracil because the dose of 5-fluorouracil is too high, owing to its reduced degradation by dihydropyrimidine dehydrogenase in the liver. Cancer patients being considered for 5-fluorouracil treatment are now routinely screened for dihydropyrimidine dehydrogenase deficiencies.

How does ribonucleotide reductase convert ribonucleotides into deoxyribonucleotides?

• Nucleoside 5′-diphosphates (GDP, ADP, CDP, and UDP) are converted into the corresponding deoxynucleoside 5′-diphosphates (dGDP, dADP, dCDP, and dUDP) by the enzyme ribonucleotide reductase and use of NADPH as a coenzyme.

• Ribonucleotide reductase is an ancient enzyme that is structurally similar across all species and likely played a pivotal role in converting the ancestral RNA world into the present-day DNA world. The RNA world hypothesis proposes that RNA was the first genetic molecule and that ribozymes were the biochemical catalysts for RNA and protein synthesis.

• The reduction of C-2′ on nucleoside diphosphates by nucleotide reductase requires the input of two electrons derived from NADPH that are used to reduce a pair of sulfhydryl groups in the enzyme active site. The reduction of these sulfhydryls in ribonucleotide reductase is not done by NADPH directly, but rather by a redox circuit requiring intermediary proteins (thioredoxin or glutaredoxin).

• Ribonucleotide reductase contains two subunits, R1 and R2, that function as a tetrameric complex (R1₂R2₂). The catalytic mechanism in E. coli depends on formation of a free radical to catalyze the reaction and requires contributions from a dinuclear Fe³⁺ iron center that is coordinated by an oxide ion (O²⁻).

• Substrate specificity of ribonucleotide reductase is regulated by allosteric binding of dTTP, dGTP, dATP, or ATP to the substrate specificity site in the R1 subunit. The overall activity of ribonucleotide reductase is regulated by allosteric binding of ATP and dATP to the activity site, such that ATP is an activator and dATP an inhibitor.

How is thymidylate synthesis targeted by anticancer drugs?

• Thymine is a pyrimidine base formed by methylation of uracil on C-5 in a reaction catalyzed by the enzyme thymidylate synthase, which converts dUMP (deoxyuridylate) to dTMP (thymidylate) and uses the coenzyme N⁵,N¹⁰-methylenetetrahydrofolate (N⁵,N¹⁰-methylene-THF).

• dUMP is derived from three sources in E. coli cells: (1) dephosphorylation of dUTP by the enzyme dUTP diphosphohydrolase; (2) deamination of dCTP by dCTP deaminase to generate dUTP, which is then converted to dUMP by dUTP diphosphohydrolase; or (3) phosphorylation of deoxyuridine by the enzyme thymidine kinase to yield dUMP.

• Thymidylate synthesis can be disrupted by two mechanisms: (1) direct inhibition of thymidylate synthase by 5-fluoro-2′-deoxyuridine-5′-monophosphate or by folate analogs such as raltitrexed; or (2) indirect inhibition of thymidylate synthase by using the folate analog methotrexate, which prevents regeneration of N⁵,N¹⁰-methylene-THF by inhibiting dihydrofolate reductase activity.

• Three mechanisms are known to modify the effectiveness of antifolate medications: (1) point mutations in the dihydrofolate reductase coding sequence to lower methotrexate binding affinity, (2) dihydrofolate reductase gene amplification to increase expression of the dihydrofolate enzyme, and (3) overexpression of the multidrug resistance protein that rapidly exports methotrexate from the cell.

• In most cancer therapies, patients are treated with a “cocktail” of anticancer drugs, which can include inhibitors of the multidrug resistance protein, as well as inhibitors of enzymes required for DNA synthesis and cell growth and antibodies that target membrane proteins to initiate the cell death pathway.

Everyday example: CO-21: Methotrexate-Resistant Cancer