Nucleotide Metabolism

What are the biological functions of purine and pyrimidine 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.

● 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.

What reactions are required for purine synthesis and how do defects in purine metabolism cause gout and Lesch-Nyhan disease?

● 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 carbons come from glycine (C-4 and C-5), HCO₃^– (C-6), and two molecules of N₁₀-formyltetrahydrofolate (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 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.

● Gout is caused by the buildup of uric acid crystals (sodium urate) in the joints and kidneys. The big toe is a common joint affected by uric acid because of poor circulation in the foot and the frequency of blunt injury, which releases uric acid crystals into the synovial fluid.

● 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.

What reactions are required for pyrimidine synthesis and degradation?

● 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.

● 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 pyrimidine nucleotides UMP, CMP, and deoxythymidine-5’-monophosphate (dTMP) are degraded by a common three-reaction pathway converting uracil and thymine into β-alanine and β-aminoisobutyrate, respectively.

What reactions are required for deoxyribonucleotide synthesis and how do some chemotherapeutic cancer drugs disrupt DNA synthesis?

● 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 using NADPH as a coenzyme.

● The reduction of C-29 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 is unusual in that it 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.

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