Biochemistry Topics   

Nucleotides

Structure and Nomeclature

Bases are the nitrogenated components of nucleic acids and may be purines (two rings) or pyrimidines (one ring).

Adenine and guanine are the purines in nucleic acids.

Thymine, cytosine and uracil are the pyrimidines in nucleic acids.

Other bases that are not part of nucleic acids but have important metabolic roles include orotate, dihydroorotate, hypoxanthine and xanthine.

Nucleosides are bases linked to a ribose or deoxyribose at the sugar's 1' position.

Nucleotides, the building blocks of nucleic acids, are nucleoside phosphates, with the phosphodiester bond at the sugar's 5' position.

Ribonucleotides are synthesized first, then deoxyribonucleotide are synthesized from modified ribonucleotides.

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De Novo Purine Synthesis

The purine nucleotides used in nucleic acid synthesis are derived from inositate (IMP), which is formed in a ten step pathway from 5-phosphoribosyl-1-pyrophosphate (PRPP). PRPP is formed from ribose 5-phosphate (from the pentose pathway) by PRPP synthetase.

The formation of IMP starts with the addition of an amino group from glutamine to the 1' position of ribose, which will become the N-9 of the purine ring. This is the committed step of the pathway.

In subsequent steps, ring sections are added by glycine, 10-formyltetrahydrofolate, another glutamine, carbon dioxide, aspartate and another 10-formyltetrahydrofolate.

The de novo synthesis of purines is regulated by feedback inhibition. Adenylate (AMP), guanylate (GMP) and IMP are allosteric inhibitors of PRPP synthetase and the initial amination of PRPP by glutamine.

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De Novo Pyrimidine Synthesis

The pyrimidine nucleotides used in nucleic acid synthesis are derived from orotidylate (OMP), which is formed from the conjugation of orotate with PRPP by a transferase.

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Orotate is synthesized from carbamoyl phosphate and aspartate. The committed step in the de novo synthesis of pyrimidines is the formation of N-carbamoyl aspartate catalyzed by aspartate transcarbamoylase.

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Two more reactions are required to produce orotate. The enzymes that catalyze these last tree reactions are part of a complex known as CAD.

Carbamoyl phosphate synthase II provides the carbamoyl phosphate needed for pyrimidine synthesis in the cytosol (another carbamoyl phosphate synthase I works for the urea cycle).

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De novo pyrimidine synthesis is regulated by feedback inhibition. Uridylate (UMP) inhibits the formation of carbamoyl phosphate by carbamoyl phosphate synthase II. Cytidylate inhibits the formation of N-carbamoylaspartate by aspartate transcarbamoylase (the committed step of the pathway).

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Inosinate (IMP) is converted to adenylate (AMP) or guanylate (GMP) in separate two-step pathways. The conversion of AMP requires aspartate.

IMP + Asp + GTP ---> AMP + fumarate + GDP + Pi

The conversion to GMP requires NAD+, ATP  and glutamine.

IMP + Gln + NAD+  + ATP ---> GMP + Glu + NADH + H+  + AMP + PPi

Purine nucleotides can also be synthesized from free purine bases derived from by nucleic acid degradation. This occurs by a salvage pathway which is much less costly than the de novo pathway  and decreases the amount of uric acid produced by purine degradation.

To salvage degradation purines, they receive a ribose phosphate moiety from PRPP. The transfer to adenine is catalyzed by adenine phosphoribosyl transferase, whereas hypoxanthine-guanine phosphoribosyl transferase catalyzes the formation of inosinate and guanylate.

Both purine and pyrimidine monophosphates are converted to diphosphates by specific nucleotide monophosphate kinases using ATP.

GMP + ATP ---> GDP + ADP

Diphosphates and triphosphates are interconverted by nucleoside diphosphate kinase, a broad specificity enzyme.

GDP + ATP ---> GTP + ADP

AMP, ADP and ATP are interconverted by adenylate kinase.

AMP + ATP ---> 2ADP

Orotidylate is decarboxylated to uridylate (UMP).

OMP + H2O ---> UMP + HCO3-

As described above, UMP is converted to the diphosphate by UMP kinase and to the triphosphate by nucleoside diphosphate kinase. UTP is converted to cytidylate triphosphate (CTP) in a reaction that requires glutamine.

UTP + Gln + ATP + H2O  --->  CTP + Glu + ADP + Pi

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Ribonucleotide reductase catalyzes the final stage in the synthesis of deoxyribonucleotides from ribonucleotides by replacing the 2' hydroxyl by a hydrogen.

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The overall pathway is a complex. Electrons from NADPH are transferred to the substrate through a series of carriers:

Ribonucleotide reductase consists of two subunits. One contains a substrate binding site, two allosteric control sites and a sulfhydryl pair (R(SH)2 ). The other contains two sites were free radicals are generated by iron atoms (OFe2 ). The sulfhydryl and free radical sites together form the overall catalytic site.

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Thymidylate synthase catalyzes the methylation of deoxyuridylate (dUMP) to yield deoxythymidilate (dTMP) using a tetrahydrofolate derivative as a methyl donor.

dUMP + methylene tetrahydrofolate → dTMP + dihydrofolate

Tetrahydrofolate is regenerated by catalysis of dihydrofolate reductase.

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Nucleotides are hydrolytically degraded to nucleosides by nucleotidases. Phosphorolytic cleavage of nucleosides to free bases and ribose (or deoxyribose) 1-phosphate is catabolized by nucleoside phosphorylase

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Adenylate is deaminated to inositate by adenylate deaminase before removing the phosphate and base.

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Guanine is deaminated by guanase after the ribose and phosphate have been removed by nucleotidase and nucleoside phosphorylase, yielding xanthine. Hypoxanthine is also converted to xanthine by xanthine dehydrogenase.

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Xanthine is converted to uric acid for excretion by xanthine oxidase.

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There is no similar degradation for pyrimidines because they are precursors of citric acid cycle intermediates.

cytosine --->  uracil --->  acetyl CoA

thymine --->  succinyl CoA

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Some chemotherapeutic agents have an effect on nucleotide metabolism. Flourouracil is converted in vivo into F-dUMP, and analog of deoxyuridylate (dUMP), that irreversibly inhibits thyamidylate synthase after acting as substrate (suicide inhibition).

The synthesis of dTMP can also be blocked by inhibiting the regeneration of tetrahydrofolate. Analogs of dihydrofolate such as methotrexate are potential inhibitors of dihydrofolate reductase.

There are three main diseases related to nucleotide metabolism: adenosine deaminase deficiency (ADD), gout and Lesh-Nyban syndrome. ADD results in immunodeficiency ("bubble boy" syndrome).

Gout occurs when uric acid precipitates in joints causing an inflammatory reaction. Although the cause in most cases is unknown, a small portion of patients has a deficiency of hypoxanthine-guanine phosphoribosyl transferase, a salvage pathway enzyme. This accelerates de novo purine synthesis and degradation.

Allopurinol, an inhibitor of xanthine oxidase, is used to treat gout. Xanthine oxidase hydrolyzes allopurinol to alloxanthine, which remains tightly bound to the active site. As a result, concentrations of hypoxanthine and xanthine increase.

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A total absence of hypoxanthine-guanine phosphoribosyl transferase leads to increased PRPP levels that cause Lesh-Nyban syndrome, a neurological disorder characterized by compulsive self-destructive behavior.

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