BIOCHEM

• 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)
• Nucleotide bases, nucleosides, and nucleotides:

  • Base: Base
  • Base + Sugar = Nucleoside
  • Base + Sugar + Phosphate = Nucleotide
  • Example: Adenine - Deoxyadenosine - Deoxyadenosine 5’-triphosphate (dATP)
• Purine Bases:

  • Adenine, Guanine
  • Hypoxanthine, Xanthine
  • Dimethylaminoadenine, 7-Methylguanine
  • 1,3,7-trimethylxanthine, 3,7-dimethylxanthine
  • Methylxanthines present in foods
  • Structures of purine bases
• Pyrimidine Bases:

  • Cytosine, Thymine, Uracil
  • 5-Methylcytosine, 5-Hydroxymethylcytocine
  • Uridine, Cytidine
  • Guanosine, Adenosine
  • Structures of pyrimidine bases
• Esterification:

  • Esterification by the same phosphate of a second -OH of the same sugar from cyclic phosphodiesters is involved in secondary messenger functions
• Nucleic Acid Nomenclature:

  • Base: Nucleoside: Nucleotide: Nucleic Acid
  • Examples: Adenine (A): Adenosine: Adenylate: RNA
  • Guanine (G): Guanosine: Guanylate: DNA
  • Cytosine (C): Cytidine: Cytidylate: RNA
  • Thymine (T): Deoxythymidine: Deoxythymidylate: DNA
  • Uracil (U): Uridine: Uridylate: RNA
• Nucleotides synthesis:

  • De novo biosynthesis pathways start from simple precursors like 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
• Purine and Pyrimidine synthesis:

  • Two means of synthesis: de novo (from bits and parts) and salvage (recycle from pre-existing nucleotides)
  • Precursors involved in de novo purine biosynthesis include amide Ns of glutamine, carbon from formate, C=C-N from glycine, carbon from CO2, and amino N from aspartate
• 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
• Steps in nucleotide synthesis:

  • Many steps require an activated ribose sugar (PRPP) for synthesis
  • Committed step is the point of no return and often regulated by final product through feedback inhibition
• Summary of IMP Synthesis:

  • The purine ring is built stepwise onto the ribose backbone
  • Purine ring synthesis requires 5 synthetase activities, 2 transamidations, 2 formylations, 1 carboxylation, and 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 the sequential removal of bits and pieces, with the end product being uric acid
• Excess uric acid can cause gout, characterized by acute arthritic joint inflammation
• 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
• 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 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
  • 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:

  • 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 characterized by a defect in de novo synthesis of pyrimidines, resulting in 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): Restores depleted UMP levels and downregulates the pathway via feedback inhibition, leading to less orotate production
• Biosynthesis: Purine vs Pyrimidine:

  • Purines are synthesized on PRPP and regulated by GTP/ATP, generating IMP
  • Pyrimidines are synthesized separately and added to PRPP, regulated by UTP, generating UMP/CMP
• 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, essential for DNA and RNA synthesis
  • 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, leading to decreased THF for one-carbon unit transfer
  • 5-fluorouracil, an analogue of dUMP, inhibits thymidylate synthetase, affecting dTMP synthesis
• Concepts from Today’s Lecture:

  • Nucleotides can be synthesized through de novo and salvage pathways
  • Pathways are regulated by feedback inhibition
  • Specific degradation pathways exist for pyrimidines
  • Molecular basis of metabolic diseases was discussed