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Nucleotide Metabolism

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I. Overview

• Building blocks for DNA and RNA synthesis
• Energy currency (ATP, GTP)
• Second messengers (cAMP, cGMP)
• Activated carriers in biosynthesis (UDP-glucose, CDP-choline)
• Coenzyme components (NAD, FAD, CoA)
• Allosteric regulators of metabolic enzymes

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II. Structure of Nucleotides

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III. Purine Synthesis

A. De Novo Purine Synthesis

• Starting material: 5-phosphoribosyl-1-pyrophosphate (PRPP) - formed from ribose 5-phosphate + ATP (by PRPP synthetase)
• Requires 6 ATP per purine synthesized
• First purine product: Inosine monophosphate (IMP)
• AMP and GMP are each derived from IMP in 2-step reactions

B. IMP → AMP and IMP → GMP

• IMP → AMP: requires aspartate, GTP (energy); 2 steps
• IMP → GMP: requires NAD⁺ oxidation then glutamine amination, uses ATP (energy)
• Cross-regulation: GTP is used to make AMP; ATP is used to make GMP (balances production)

C. Purine Salvage Pathway

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IV. Purine Degradation and Clinical Disorders

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V. Pyrimidine Synthesis

1. Carbamoyl phosphate synthesized by CPS II (cytosolic) using glutamine + CO₂ + 2ATP (note: CPS I is mitochondrial and used in urea cycle)
2. Carbamoyl phosphate + aspartate → dihydroorotate → orotate
3. Orotate + PRPP → Orotidine 5'-monophosphate (OMP) → UMP
4. UMP → UTP → CTP (via CTP synthetase)
5. dTMP synthesis: UDP → dUDP → dUMP → dTMP (by thymidylate synthase using N5,N10-methylene-THF)

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VI. Deoxyribonucleotide Synthesis

• Contains two subunits: R1 (α, regulatory) and R2 (β, contains tyrosyl radical)
• Reducing equivalents from thioredoxin → NADPH via thioredoxin reductase (a selenoprotein)
• Allosteric regulation is complex:
  • Activity site on R1: dATP inhibits (overall activity); ATP activates
  • Specificity site on R1: controls which substrate is used

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VII. Clinical Disorders of Nucleotide Metabolism

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VIII. Pharmacological Targets in Nucleotide Metabolism

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Quick Summary Table: De Novo Synthesis Comparison

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De Novo Synthesis of Nucleotides

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PART 1: De Novo Purine Synthesis

Overview

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Step-by-Step: The 11 Reactions to IMP

Precursor: PRPP Formation (not the committed step)

• Enzyme: PRPP synthetase
• Ribose 5-phosphate comes from the pentose phosphate pathway
• PRPP is a shared substrate used in both purine synthesis, pyrimidine synthesis, and salvage pathways

Step 1 - COMMITTED STEP

• Enzyme: Glutamine phosphoribosylpyrophosphate amidotransferase (GPAT)
• This is the first committed, rate-limiting step
• Incorporates N-9 of the purine ring (from glutamine's amide nitrogen)
• The enzyme is a monomer (active) ↔ dimer (inactive) interconversion
• Strongly inhibited by AMP and GMP (end-product feedback inhibition at distinct allosteric sites)

Steps 2-10: Building the Ring Atom by Atom

Memory tip for purine ring atoms: "A Genuine Couple Go Alone"

• Aspartate → N1
• Glycine → C4, C5, N7
• CO₂ → C6
• Glutamine → N3, N9
• Alone = N¹⁰-formyl-THF → C2, C8

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IMP → AMP (2 steps)

1. Adenylosuccinate synthetase: IMP + Aspartate + GTP → Adenylosuccinate + GDP + Pi
   • Note: GTP is the energy source (not ATP)
2. Adenylosuccinate lyase: Adenylosuccinate → AMP + Fumarate
   • Aspartate donates its nitrogen; its carbons leave as fumarate (identical mechanism to urea cycle's argininosuccinate → arginine + fumarate)

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IMP → GMP (2 steps)

1. IMP dehydrogenase: IMP + NAD⁺ → XMP (xanthosine monophosphate) + NADH
   • Oxidation of hypoxanthine base to xanthine
2. GMP synthetase: XMP + Glutamine + ATP → GMP + Glutamate + AMP + PPi
   • Glutamine donates its amide nitrogen to form the 2-amino group of guanine

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Regulation of De Novo Purine Synthesis

                      AMP ──┐
                             ├──► Inhibit GPAT (committed step)
                      GMP ──┘
                      
                      AMP ──► Inhibit adenylosuccinate synthetase (IMP→AMP branch)
                      GMP ──► Inhibit IMP dehydrogenase (IMP→GMP branch)
                      
                      ATP ──► needed to make GMP (cross-activation)
                      GTP ──► needed to make AMP (cross-activation)

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PART 2: De Novo Pyrimidine Synthesis

Overview

Origin of Pyrimidine Ring Atoms

Only 3 precursor molecules needed: CO₂ + Glutamine + Aspartate (via carbamoyl phosphate)

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Step-by-Step: UMP Synthesis (6 steps)

Step 1 - COMMITTED STEP

• Enzyme: Carbamoyl Phosphate Synthetase II (CPS II) - cytosolic
• This is the committed, rate-limiting step of pyrimidine synthesis
• CPS II is distinct from CPS I (mitochondrial, urea cycle) - different location, different substrate (glutamine vs NH₃), different regulation
• CPS II is part of the CAD trifunctional enzyme

Step 2

• Enzyme: Aspartate transcarbamoylase (part of CAD complex)

Step 3

• Enzyme: Dihydroorotase (part of CAD complex)

CAD = CPS II + Aspartate transcarbamoylase + Dihydroorotase (one polypeptide, 3 activities)

Step 4

• Enzyme: Dihydroorotate dehydrogenase - located on the inner mitochondrial membrane (the only mitochondrial step)
• Uses NAD⁺ as electron acceptor

Step 5

• Enzyme: Orotate phosphoribosyltransferase (part of UMP synthase bifunctional enzyme)
• PRPP attaches the ribose-5-phosphate to the base

Step 6

• Enzyme: Orotidylic acid decarboxylase (OMP decarboxylase) (part of UMP synthase)

UMP synthase = Orotate phosphoribosyltransferase + OMP decarboxylase (bifunctional enzyme; deficiency causes hereditary orotic aciduria)

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UMP → Other Pyrimidines

UMP → UDP → UTP → RNA
                 ↓
           CTP synthetase (+Glutamine, +ATP)
                 ↓
               CTP → RNA

UDP → dUDP → dUMP → dTMP (via thymidylate synthase + N⁵,N¹⁰-methylene-THF)
                             → dTDP → dTTP → DNA

• Thymidylate synthase: dUMP + N⁵,N¹⁰-methylene-THF → dTMP + DHF
• DHF is recycled back to THF by dihydrofolate reductase (DHFR)
• Both enzymes are major drug targets (5-FU inhibits thymidylate synthase; methotrexate inhibits DHFR)

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Regulation of De Novo Pyrimidine Synthesis

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PART 3: Comparison Summary

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Key Pharmacological Targets

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