Nucleotide Metabolism

• What are the three different types of nucleotide metabolism?

  De novo synthesis, Salvage pathway, Nucleotide degradation
• What percentage of nucleotide supply is provided by each of the following?

  Dietary: 30%, De novo synthesis in tissues: 70%
• What is the process of pyrimidine synthesis?

  Ring synthesized first, then attached to sugar
• What is the process of purine synthesis?

  Ring synthesized off sugar
• What is the common intermediate of pyrimidine synthesis?

  Uridylate (UMP)
• What is the common intermediate of purine synthesis?

  Inosinate (IMP)
• How is ribose 5-phosphate converted to PRPP?

  Using ATP (attachment of PPi) via PRPP synthetase
• What is the starting material for purine synthesis?

  5-Phosphoribosyl-1-amine
• How is PRPP converted to 5-Phosphoribosyl-1-amine?

  Using NH3 from Gln (hydrolysis) displacing PPi
• What enzyme is involved in the production of Carbamoyl Phosphate?

  Carbamoyl phosphate synthetase
• What are the 3 steps involved in the production of Carbamoyl Phosphate and what do they each require?

  1. Bicarbonate → Carboxyphosphate (requires 1 ATP)
  2. Carboxyphosphate → Carbamic Acid (requires NH3 from Gln)
  3. Carbamic Acid → Carbamoyl phosphate (requires 1 ATP)
• What enzyme is involved in the conversion of Carbamoyl phosphate to Carbamoylaspartate and what does it require?

  Aspartate transcarbamoylase aka ATCase (requires Asp)
• What steps are involved in the pyrimidine ring formation and what do they require?

  1. Carbamoylaspartate → Dihydroorotate
  2. Dihydroorotate → Orotate (CoQ or NAD+)
• What type of reaction is Carbamoylaspartate → Dihydroorotate and what does it form?

  Dehydration; forms water
• What type of reaction is Dihydroorotate → Orotate and what happens to the cofactor?

  Oxidation; cofactor reduced
• What steps are involved in the conversion of Orotate to UMP and what do they require?

  1. Orotate + PRPP → Orotidylate (requires 2 ATP)
  2. Orotidylate → Uridylate
• What type of reaction is Orotate + PRPP → Orotidylate and what enzyme is required?

  Coupling reaction; Orotate phosphoribosyltransferase
• What type of reaction is Orotidylate → Uridylate and what enzyme is required?

  Decarboxylation reaction; Orotidylate decarboxylase
• How is UMP converted to UTP, what is required and what enzymes are involved?

  1. UMP → UDP (requires 1 ATP); via Uridylate kinase
  2. UDP → UTP (requires 1 N*TP); via Non-specific kinase
• How is CTP made – what is required, what enzyme is involved, and what type of reaction is?

  From UTP, requires 1 ATP and NH3 from Gln; via Cytidylate synthetase; Amination reaction
• What is the feedback regulation of ATCase?

  CTP inhibits ATCase

  ATP stimulates ATCase
• What atoms does each cofactor contribute to pyrimidine structure during synthesis?

  Bicarbonate → C2

  Glutamine → N3

  Aspartate → N1, C4-6
• What are the 3 sites of Carbamoyl Phosphate Synthetase?

  1. Glutamine hydrolysis site
  2. Bicarbonate phosphorylation site
  3. Carbamic acid phosphorylation site
• What are the 4 actions of Carbamoyl Phosphate Synthetase?

  1. Phosphorylate bicarbonate using ATP
  2. Hydrolyze NH3 from glutamine
  3. Add NH3 to Carboxyphosphate
  4. Transfer phosphate from ATP onto Carbamic acid to form Carbamoyl Phosphate
• What are deoxyribonucleotides made from, what enzyme is involved, and what is used?

  Made from nucleoside diphosphates; via Ribonucleotide reductase; uses NADPH
• What is the mechanism of Ribonucleotide reductase in forming deoxyribonucleotides?

  1. Iron cluster (β2 subunit of R2 dimer) gives up an electron (free radical) to a key tyrosine residue
  2. Free radical transfer from Tyr radical to Cys residue (R1 domain)
  3. Cys radical transfers to C3’ of bound nucleotide (active site)
  4. Radical on C3’ promotes release of C2’ OH as water

  • Also leads to formation of disulfide bond between enzyme Cys residues

  1. Free radical passed back to the R2 domain in exchange for a proton
  2. Disulfide bond reduced using NADPH to regenerate the enzyme

  • Via Thioredoxin reductase
• What are the 3 sites of regulation of ribonucleotide reductase?

  1. Active site
  2. Activity (determining) site
  3. Specificity (determining) site
• What is the regulation of catalysis at the activity site of ribonucleotide reductase?

  Stimulated by ATP

  Inhibited by dATP
• What is the regulation of pyrimidine reduction at the specificity site of ribonucleotide reductase?

  Stimulated by dATP or ATP

  Inhibited by TTP
• What is the regulation of GDP reduction at the specificity site of ribonucleotide reductase?

  Stimulated by TTP
• What is the regulation of ADP reduction at the activity site of ribonucleotide reductase?

  Stimulated by dGTP
• What is the purpose of ribonucleotide reductase regulation?

  To ensure a balance of all nucleotides
• What is the process of making Thymine, what does it require, and what enzymes are used in each step?

  1. dUTP → dUMP (via dUTP pyrophosphatase)
  2. dUMP → (d)TMP (via Thymidylate synthase; uses tetrahydrofolate derivative)
  3. TMP → TTP
• What is the problem in thymine synthesis and what is the solution?

  DNA polymerase cannot distinguish between TTP and dUTP; quick conversion to dUMP
• What is the mechanism of thymine synthesis?

  1. Sulfhydryl group of thymidylate synthase attacks C6 of dUMP
  2. Proton at C5 abstracted by S of enzyme
  3. Hydride and CH2 transferred from tetrahydrofolate derivative to C5
  4. Enzyme released
• What is the rate limiting step in DNA synthesis?

  Thymidylate synthase
• What atom/s does the first glutamine contribute in purine synthesis and what cofactor is required?

  N9; requires H2O
• What atom/s does glycine contribute in purine synthesis and what cofactor is required?

  C4, C5, N7; requires ATP
• What atom/s does the first tetrahydrofolate contribute in purine synthesis and what cofactor is required?

  C8, requires no cofactor
• What atom/s does the second glutamine contribute in purine synthesis and what cofactor is required?

  N3; requires ATP and H2O