CHAPTER 33

• Functional roles of nucleotides:

  • Precursors of DNA and RNA
  • Carrier of chemical energy (ATP, GTP)
  • Second messengers (cAMP, cGMP)
  • Components of activated biosynthetic intermediates (UDP-glucose, CDP-diacylglycerol)
  • Components of cofactors (NAD, FAD, S-adenosylmethionine, Coenzyme A)
• Purine Bases:

  • Adenine
  • Guanine
  • Hypoxanthine
  • Xanthine
  • Dimethylaminoadenine
  • 7-Methylguanine
  • 1,3,7-trimethylxanthine
  • 3,7-dimethylxanthine
• Pyrimidine Bases:

  • Cytosine
  • Thymine
  • Uracil
  • 5-Methylcytosine
  • 5-Hydroxymethylcytocine
  • Uridine
  • Cytidine
• Esterification:

  • By the same phosphate of a second -OH of the same sugar from cyclic phosphodiesters
  • Secondary messenger
• Nucleic Acid Nomenclature:

  • Purine: Adenine (A), Guanine (G)
  • Pyrimidine: Cytosine (C), Thymine (T), Uracil (U)
• Purines and Pyrimidines Synthesis:

  • De novo and salvage pathways
  • Degradation
  • Relevant disease states
  • Relevant clinical applications
• Where do nucleotides come from?

  • De novo biosynthesis pathways start from simple precursors (CO2, NH3, amino acids, ribose-5-phosphate)
  • Purine ring is built up one or a few atoms at a time
  • Pyrimidine is built from orotate
  • Salvage pathways involve recycling of free bases and nucleosides obtained from nucleic acid breakdown
• Synthesis Pathway:

  • For both purines and pyrimidines, there are de novo (from bits and parts) and salvage (recycle from pre-existing nucleotides) pathways
• Purine nucleotide synthesis:

  • Carbohydrate: Ribose
  • Amino acid precursor: glycine
  • Nitrogen donors: glutamine, aspartate
• Pyrimidine nucleotide synthesis:

  • Carbohydrate: Ribose
  • Amino acid precursor: aspartate
  • Nitrogen donors: glutamine
• Purine Biosynthesis (de novo):

  • Atoms derived from aspartic acid, glycine, glutamine, CO2, tetrahydrofolate
  • Requires 4 ATPs
  • Purines are synthesized on the Ribose ring
  • Committed step is inhibited by AMP, GMP, IMP
• Summary of IMP Synthesis:

  • The purine ring is built stepwise onto the ribose backbone
  • Purine ring synthesis requires 5 synthetase activities (5 ATPs used), 2 transamidations, 2 formylations, 1 carboxylation, 2 cycli
• Purine ring synthesis requires:

  • 5 synthetase activities (5 ATPs used)
  • 2 transamidations
  • 2 formylations
  • 1 carboxylation
  • 2 cyclizations
  • 1 "de facto" transamination
• AMP synthesis:

  • Amination of the purine ring occurs in two steps
  • Intermediate: adenylosuccinate
  • Adenylosuccinate synthetase requires GTP
• GMP synthesis:

  • Oxidation creates a reactive carbonyl group
  • Amination of the purine ring via transamidation
  • Intermediate: xanthylate (XMP)
  • XMP glutamine amidotransferase requires ATP
• Purine degradation involves:

  • Sequential removal of bits and pieces
  • End product is uric acid
  • Uric acid is primate-specific
  • Excreted in urine
• Excess uric acid causes gout:

  • Primary gout (hyperuricemia)
  • Inborn errors of metabolism that lead to overproduction of uric acid
  • Overactive de novo synthesis pathway
  • Leads to deposits of uric acid in the joints
  • Causes acute arthritic joint inflammation
  • Offal foods such as liver, kidneys, tripe, sweetbreads, and tongue should be avoided
• Salvage pathway for purines:

  • Hypoxanthine or guanine + PRPP = IMP or GMP + PPi
  • Enzymes involved: HGPRTase and APRTase
• Hypouricemia and increased excretion of hypoxanthine and xanthine are associated with xanthine oxidase deficiency, due to a genetic defect or severe liver damage
• Adenosine deaminase deficiency:

  • T cells and B cells are sparse and dysfunctional
  • Patients suffer from severe immunodeficiency
  • Infants often succumb to fatal infections
• Purine nucleoside phosphorylase deficiency:

  • Results in severe T cell deficiency
  • Normal B cell function
  • Immune dysfunctions appear to result from accumulation of dGTP and dATP
• Purine deficiency states are rare in humans and are associated with primary deficiencies of folic acid
• Biosynthesis of pyrimidines:

  • Pyrimidine rings are synthesized independent of the ribose and transferred to the PRPP (ribose)
  • Generated as UMP (uridine 5’-monophosphate)
  • Synthesized from glutamine, CO2, aspartic acid
  • Requires ATP
• Metabolism of pyrimidine nucleotides:

  • Carbamoyl phosphate synthetase II (gln) is involved in the synthesis
  • Classic feedback regulation is in place to prevent trapping and promote balance of purines and pyrimidines
• Regulation of pyrimidine biosynthesis occurs at the first step in the pathway (committed step)
• Regulation of Pyrimidine Biosynthesis:

  • Regulation occurs at the first step in the pathway (committed step): 2ATP + CO2 + Glutamine = carbamoyl phosphate
  • This step is inhibited by UTP
  • Feedback Inhibition: If there is an abundance of UTP, the pathway is downregulated to prevent overproduction
• Clinical Disorders of Pyrimidine Metabolism:

  • End products of pyrimidine metabolism are carbon dioxide, ammonia, beta-alanine, and beta-anisobutyrate
  • These compounds are highly water-soluble, allowing for easy excretion through urine
  • Hereditary Orotic Aciduria is a rare disorder caused by a defect in de novo synthesis of pyrimidines, leading to severe anemia and growth retardation
  • Treatment involves feeding UMP to restore depleted levels
• Why does UMP Cure Orotic Aciduria?

  • Disease (-UMP): No UMP or excess orotate
  • Disease (+UMP): Restore depleted UMP levels and downregulate the pathway via feedback inhibition
  • This process involves Carbamoyl Phosphate, UMP, Orotate, UMP Synthetase, and UTP with feedback inhibition
• Biosynthesis: Purine vs Pyrimidine:

  • Purines and pyrimidines are synthesized on PRPP
  • Purine biosynthesis is regulated by GTP/ATP, generates IMP, and requires energy
  • Pyrimidine biosynthesis is regulated by UTP, generates UMP/CMP, and requires energy
• Pyrimidine Degradation/Salvage:

  • Pyrimidine rings can be fully degraded to soluble structures and can also be salvaged by reactions with PRPP
  • Degradation pathways for purines and pyrimidines are distinct, but salvage pathways are similar
• Conversion of Ribonucleotides to Deoxyribonucleotides:

  • Specific kinases convert NMP to NDP
  • Ribonucleotides are converted to deoxyribonucleotides by ribonucleotide reductase
• Ribonucleotide Reductase:

  • Catalyzes the conversion of NDP to dNDP
  • Highly regulated enzyme that controls the level of cellular dNTPs
  • Activated prior to DNA synthesis and controlled by feedback inhibition
• Treatment of Certain Cancers:

  • Anticancer drugs inhibit the biosynthesis of purine and pyrimidine, suppressing cancer cell growth
  • These drugs slow down DNA synthesis and cell growth, affecting both cancer and normal cells
• Action of Anti-Cancer Drugs:

  • Glutamine antagonists like azaserine inhibit CPS II in pyrimidine synthesis and reaction 2 in purine synthesis
  • Folate antagonists like methotrexate inhibit DHF reductase, affecting dTMP synthesis and purine synthesis
  • 5-fluorouracil, an analogue of dUMP, inhibits thymidylate synthetase, affecting dTMP synthesis
• Concepts from Today’s Lecture:

  • Nucleotides can be made through de novo and salvage pathways
  • Pathways are regulated by feedback inhibition
  • Specific degradation pathways exist for pyrimidines
  • Understanding the molecular basis of metabolic diseases mentioned in the lecture