Nucleotide Biosynthesis, Salvage Pathways, and Regulation

Structure and Properties of Nucleotides

Nucleotides (phosphoric esters of nucleosides: nucleoside + phosphate = pentose phosphate linked at C-5) yield nucleosides upon dephosphorylation (nitrogenous base [NB] + pentose via glycosidic linkage).

Nitrogenous Bases and Pentoses

• Pyrimidines (joined via N-1): Cytosine (C), Uracil (U), Thymine (T)
• Purines (joined via N-9, double ring): Adenine (A), Guanine (G)
• Properties of Nitrogenous Bases (NB): Absorption in the UV spectrum (260 nm), weak basic character, planar structures, ability to form hydrogen bonds, and tautomerism (keto-enol and amine-imine).
• Pentose: May be either D-ribose (RNA) or 2-deoxy-D-ribose (DNA).

De Novo Nucleotide Biosynthesis

Origin of Atoms:

• Purines: CO₂, Glycine (Gly), Aspartate (Asp), Glutamine (Gln), and Tetrahydrofolate (THF).
• Pyrimidines: CO₂, Glutamine (Gln), and Aspartate (Asp).

Common Pathway: PRPP Synthesis

Ribose 5-phosphate (from pentose phosphate pathway) + ATP → 5-Phosphoribosyl-1-pyrophosphate (PRPP) + AMP (catalyzed by PRPP synthetase).

De Novo Purine Biosynthesis

Occurs in the cytosol; it is a lengthy and energetically expensive pathway where the purine ring is assembled directly on the ribose backbone:

1. PRPP + Glutamine + H₂O → 5-Phosphoribosylamine (PRA) + Glutamate + PPi (via Glutamine-PRPP amidotransferase)
2. PRA + Glycine + ATP → … + ADP
3. … + N¹⁰-Formyl-THF → … + THF
4. … + Glutamine + ATP → … + Glutamate + ADP
5. … + ATP → Ring cyclization + ADP
6. … + CO₂ → …
7. … + Aspartate + ATP → … + ADP
8. Series of reactions → Release of Fumarate
9. … + N¹⁰-Formyl-THF → … + THF
10. Loss of H₂O → Inosine Monophosphate (IMP) (the purine base hypoxanthine is established within IMP)

Branching from IMP:

• Synthesis of AMP: IMP + Aspartate + GTP → Adenylosuccinate + GDP + Pi (via adenylosuccinate synthetase) → AMP + Fumarate (via adenylosuccinate lyase).
• Synthesis of GMP: IMP + H₂O + NAD⁺ → Xanthosine Monophosphate (XMP) + NADH + H⁺ (via IMP dehydrogenase); XMP + Glutamine + ATP + H₂O → GMP + Glutamate + AMP + PPi (via GMP synthase).

Note: Nucleoside monophosphate kinases are base-specific, whereas nucleoside diphosphate kinase (NDPK) is non-specific.

Regulation of Purine Biosynthesis

• Ribose-5-phosphate / PRPP Synthetase: Partial feedforward/feedback inhibition by AMP, GMP, and IMP.
• Glutamine-PRPP Amidotransferase: Synergistic, concerted allosteric inhibition by IMP, AMP, and GMP.
• Adenylosuccinate Synthetase: Specifically inhibited by AMP.
• IMP Dehydrogenase (IMP DH): Specifically inhibited by GMP.

Purine Salvage Pathway

Recycling of nucleic acids via degradation and base salvage:

Nucleic acids → (endonuclease) → Oligonucleotides → (phosphodiesterase) → Nucleoside monophosphates (NMP) → (nucleotidase + H₂O) → Nucleosides + Pi → (nucleoside phosphorylase + Pi) → Free Nitrogenous Bases + Ribose 1-phosphate.

Reincorporation into Nucleotides:

1. Adenine + PRPP → AMP + PPi (catalyzed by adenine phosphoribosyltransferase, APRT)
2. Hypoxanthine / Guanine + PRPP → IMP / GMP + PPi (catalyzed by hypoxanthine-guanine phosphoribosyltransferase, HGPRT)

De Novo Pyrimidine Biosynthesis

Occurs in the cytosol; the pyrimidine ring is synthesized prior to attachment to ribose:

1. CO₂ + Glutamine + 2 ATP + H₂O → Carbamoyl phosphate + Glutamate + 2 ADP + Pi (via carbamoyl phosphate synthetase II [CPS II]; activated by ATP and PRPP, inhibited by CTP, UTP, and UDP)
2. Carbamoyl phosphate + Aspartate → N-Carbamoylaspartate (via aspartate transcarbamoylase, ATCase)
3. N-Carbamoylaspartate → Dihydroorotate + H₂O
4. Dihydroorotate + NAD⁺ → Orotate + NADH + H⁺ (oxidation; orotate is the complete pyrimidine base)
5. Orotate + PRPP → Orotidine Monophosphate (OMP) + PPi (via orotate phosphoribosyltransferase)
6. OMP → Uridine Monophosphate (UMP) + CO₂ (inhibited by UMP)
7. UMP + ATP → UDP + ADP (via UMP kinase)
8. UDP + ATP → UTP + ADP (via nucleoside diphosphate kinase)
9. UTP + Glutamine + ATP + H₂O → Cytidine Triphosphate (CTP) + Glutamate + ADP + Pi (via CTP synthetase; feedback inhibited by CTP)

Pyrimidine Salvage Pathway

Nucleic acids → (hydrolysis) → Uridine / Cytidine nucleosides.

• Synthesis of UMP: Uridine + ATP → UMP + ADP (via uridine kinase)
• Synthesis of CMP: Cytidine + ATP → CMP + ADP (via cytidine kinase)

Formation of Deoxyribonucleosides

1. Enzyme Action: Reduction of ribonucleotides by ribonucleotide reductase (RNR), dependent on NADPH + H⁺ → NADP⁺.
2. Electron Transport Chain 1: Mediated by thioredoxin and thioredoxin reductase.
3. Electron Transport Chain 2: Mediated by glutaredoxin and glutathione reductase.

Regulation of Ribonucleotide Reductase

• Overall Activity Regulation: Allosterically activated by ATP (turns enzyme on); allosterically inhibited by dATP (turns enzyme off).
• Substrate Specificity Regulation: Effector binding modulates active site specificity to balance relative cellular concentrations of each deoxyribonucleotide (dATP, dGTP, dCTP, and dTTP).