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L 41 Nucleotide Metabolism and Disease – Comprehensive Study Notes

I. Nucleotide structure

  • Comprised of:
    • 1 nitrogenous base (purine or pyrimidine)
    • 1 pentose monosaccharide (sugar)
    • 1–3 phosphate groups
  • Bases
    • Purines: A, G
    • Pyrimidines: C, T (DNA), C, U (RNA)
    • RNA contains uracil (U) instead of thymine (T)
    • DNA: A, G, C, T; RNA: A, G, C, U
  • Nucleoside vs. nucleotide
    • Nucleoside = pentose sugar + base
    • Nucleotide = nucleoside + phosphate(s) (-PO4)
    • Ribonucleosides (adenosine, guanosine, cytidine, uridine) have ribose → RNA
    • Deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxycytidine, deoxythymidine) have 2′-deoxyribose → DNA
    • Phosphorylation adds 1–3 phosphate groups to 5′-OH of the pentose sugar to form mono-, di-, or tri-phosphates
  • Nucleotides and nucleic acids
    • DNA/RNA are long strands of nucleotides; negative charge from –PO4^− groups forms nucleic acids
    • Ribonucleotides: ribonucleoside triphosphates (e.g., ATP,
      GTP, CTP, UTP)
    • Deoxyribonucleotides: deoxyribonucleoside triphosphates (e.g., dATP, dGTP, dCTP, dTTP)

II. Digestion of dietary nucleic acids and nutritional impact on nucleotide metabolism

  • Nutrition supports nucleotide synthesis via nitrogen and carbon from amino acids and vitamins (e.g., folic acid)
  • Dietary sources contain nucleic acids; nucleotides are denatured in the stomach and digested in the small intestine by pancreatic enzymes to nucleotides, then to nucleosides, then to free bases
  • Fate of dietary purines and pyrimidines
    • Purines: largely degraded to uric acid; uric acid enters blood and is excreted by kidneys
    • Pyrimidines: excreted in urine or salvaged for nucleotide synthesis
  • Salvage of dietary nucleic acids contributes only a minor portion of nucleotides used for DNA/RNA synthesis; most nucleotides come from de novo synthesis

III. Endogenous biosynthesis and salvage of purines (Adenosine and Guanosine)

  • Purine biosynthesis in all cells occurs by two mechanisms:
    1) De novo synthesis (primarily in liver hepatocytes in the cytoplasm)
    2) Salvage pathway (in all tissues) to reuse purine bases/nucleosides and minimize energy expenditure
  • De novo purine synthesis requires ATP and incorporation of atoms from:
    • Amino acids: Asp, Gly, Gln
    • CO2
    • N^10-formyltetrahydrofolate (folic acid derivative)
  • The salvage pathway recycles purine bases delivered from turnover or dietary nucleic acids
  • PRPP (5′-phosphoribosyl-1-p pyrophosphate) is the activated ribose donor for both de novo purine biosynthesis and salvage (and also pyrimidine biosynthesis)

IV. De novo Purine Biosynthesis

  • STEP 1: Synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP)
    • Ribose-5-phosphate (from the pentose phosphate pathway) is the ribose primer
    • PRPP synthetase transfers a pyrophosphate from ATP to C-1 of ribose-5-phosphate
    • Reaction (schematic):
      ext{Ribose-5-phosphate} + ext{ATP}
      ightarrow ext{PRPP} + ext{AMP}
    • Regulation: PRPP synthetase is inhibited by end-products of purine nucleotides (end-product inhibition)
    • PRPP serves as the activated ribose donor for both purine and pyrimidine biosynthesis and salvage
  • STEP 2 (Committed, regulatory step): Synthesis of 5′-phosphoribosylamine
    • PRPP allosterically activates Glutamine:PRPP amidotransferase (GPAT) to form 5′-phosphoribosylamine
    • End-product inhibitors: AMP and GMP inhibit GPAT when purine levels are sufficient
  • STEPS 3–11: Formation of the first purine nucleotide, IMP
    • Nine subsequent reactions (beyond the committed step) build the purine ring on the ribose-5-phosphate framework
    • Energy source: ATP is required for all steps
    • A folic acid derivative is required in two steps
    • Hypoxanthine is the base used in IMP

V. AMP and GMP synthesis from IMP; purine feedback regulation

  • IMP is the branch point for purine biosynthesis to AMP or GMP
  • AMP synthesis requires GTP and aspartate; GMP synthesis requires ATP and glutamine
  • Regulation by end-products:
    • Excess AMP inhibits AMP synthesis; pushes IMP toward GMP
    • Excess GMP inhibits GMP synthesis; pushes IMP toward AMP
    • If both AMP and GMP are high, early steps of de novo purine biosynthesis are inhibited
  • Interconversion of NMPs to NDPs and NTPs
    • Base-specific nucleoside monophosphate kinases phosphorylate NMPs to NDPs
    • Adenylate kinase is highly active in liver for ATP turnover
    • General nucleoside diphosphate kinases phosphorylate NDPs to NTPs
  • Purine salvage pathway
    • Recycles free purine bases from nucleic acid turnover or diet into nucleoside triphosphates with lower energy demand
    • Key salvage enzymes:
    • HGPRT: Hypoxanthine→IMP; Guanine→GMP (PRPP donor) (irreversible)
    • APRT: Adenine→AMP (PRPP donor) (irreversible)

VI. Synthesis of pyrimidines (thymine, cytosine, uracil)

  • Pyrimidine base is built before ribose addition (unlike purines)
  • Precursors: Glutamine, CO2, and aspartic acid supply the ring atoms; Glutamine and aspartate are used for all nucleotides
  • STEP 1: Carbamoyl phosphate synthesis is the committed regulatory step
    • CPS II catalyzes carbamoyl phosphate formation from Glutamine and CO2
    • Regulation: UTP allosterically inhibits CPS II; PRPP activates CPS II
    • Note: There are two CPS enzymes: CPS I (urea cycle in mitochondria) and CPS II (pyrimidine biosynthesis in cytosol)
  • STEPS 2–4: Synthesis of orotic acid (orotate) from carbamoyl phosphate
  • STEPS 5–6: Formation of UMP via orotate phosphoribosyltransferase activity (UMP synthase is bi-functional)
    • UMP is required for all pyrimidine nucleotides
  • UMP modification to UTP and CTP
    • UMP → UTP via kinase steps; UTP is used in RNA synthesis
    • CTP synthetase converts UTP to CTP using glutamine as amino donor; CTP used in RNA synthesis
  • Disease: Orotic aciduria is due to UMP synthase deficiency
    • Excess orotate in urine; symptoms include failure to thrive and megaloblastic anemia
    • Treatment: exogenous uridine (UTP end-product inhibition is relevant to pyrimidine biosynthesis)

VII. Pyrimidine salvage

  • Salvage of pyrimidine bases is limited; nucleosides (base + ribose) can be salvaged by nucleoside kinases to nucleotides using ATP
  • Notable point: Salvage of uridine to UTP underpins uridine-based therapy for orotic aciduria

VIII. Synthesis of deoxyribonucleotides (DNA nucleotides)

  • dNTPs are produced from ribonucleoside diphosphates (NDPs) via ribonucleotide reductase during S-phase
    • NADPH provides reducing power for the conversion of NDPs to dNDPs
    • Ribonucleotide reductase acts on both purine and pyrimidine ribonucleotides; balance of dNDPs is tightly regulated
    • Allosteric control: binding of nucleotides to ribonucleotide reductase tunes dNDP levels
  • Thymidine deoxynucleotides (dTTP) derive from dUMP
    • dUMP can be produced from UDP, dCTP, or dCMP
    • Thymidylate synthase converts dUMP to dTMP
    • N5,N10-methylene-THF donates a methyl group and is converted to DHF, which is reduced back to THF by DHF reductase
    • Phosphorylation steps: dTMP → dTDP → dTTP
    • dTTP is specifically required for DNA synthesis
  • Overall pathway outline: UMP → dCTP or dCMP → dUMP → dTMP → dTTP

IX. Catabolism and degradation of endogenous nucleotides and diseases

  • Pyrimidine catabolism: Pyrimidine ring degrades to highly soluble products (β-alanine, β-aminoisobutyrate, NH3, CO2)
  • Purine catabolism (dietary and cellular nucleotides)
    • Purines degrade to uric acid; uric acid is excreted in urine
    • Other animals further degrade uric acid to allantoin via urate oxidase (uricase); humans lack urate oxidase
  • Major steps in purine degradation:
    1) Adenosine deaminase converts AMP to IMP
    2) 5′-Nucleotidase converts AMP, IMP, GMP to their nucleoside forms
    3) Purine nucleoside phosphorylase (PNP) converts inosine and guanosine to hypoxanthine and guanine
    4) Xanthine oxidase oxidizes hypoxanthine to xanthine and then to uric acid; guanine deaminates to xanthine and is oxidized to uric acid
  • Gout: chronic hyperuricemia with recurrent inflammatory arthritis due to monosodium urate (MSU) crystals in joints

X. Hyperuricemia and gout: biochemical basis, clinical features, and treatment options

  • Hyperuricemia definition and gout risk
    • Serum uric acid (SUA) reference ranges vary by age and sex:
    • Males: 3.4-7.0~\text{mg/dL}
    • Females: 2.4-6.0~\text{mg/dL}
    • Not all individuals with hyperuricemia develop gout (about 1/3 do)
  • Causes of hyperuricemia/gout
    • Underexcretion of uric acid (>90%): kidney-related issues, age-related GFR decline, heart disease, genetic predisposition, environmental factors (drugs, lead exposure)
    • Overproduction of uric acid (less common): often idiopathic; genetic associations include Lesch-Nyhan syndrome (LNS)
  • Lesch-Nyhan syndrome (LNS)
    • X-linked recessive HGPRT deficiency
    • Inability to salvage hypoxanthine and guanine leads to excess purine degradation and uric acid production
    • Clinical features: uric acid stones, gouty arthritis, motor/cognitive/behavioral abnormalities, self-injurious behavior
  • Secondary causes of hyperuricemia
    • Chemotherapy for cancers with high cell turnover, hemolytic anemias, psoriasis, etc.
  • Gout clinical features
    • Acute attack: sudden, often nocturnal, joint swelling, warmth, redness, pain; often involves a single joint (podagra is first MTP joint in ~50% cases)
    • Chronic tophaceous gout: tophi formation, joint destruction, multiple attacks in short intervals
    • Urolithiasis: uric acid stones in kidney
  • Diagnosis
    • Definitive: visualization of needle-shaped monosodium urate crystals in synovial fluid or tophus
    • Supporting labs: inflammatory markers
  • Management and pharmacology
    • Acute gout attack (do not lower SUA during acute crisis): anti-inflammatory strategies, not SUA lowering
    • NSAIDs (e.g., indomethacin), corticosteroids, colchicine
    • Intercritical period (lower SUA to prevent crystals): uric acid lowering therapy (UALT)
    • Target SUA: <6 mg/dL
    • Initiate UALT after acute attack resolved to avoid flare-ups due to crystal resorption
    • UALT strategies
    • Uricosuric agents (increase renal excretion): Probenecid (requires adequate kidney function)
    • Agents that decrease uric acid production: Allopurinol (hypoxanthine analog) inhibits uric acid synthesis; converted to oxypurinol which inhibits xanthine oxidase; effective in both overproducers and underexcretors
    • Febuxostat: non-purine XO inhibitor; non-competitive with XO
    • Agents that convert uric acid to more soluble forms: Recombinant urate oxidase/uricase (not in humans) such as Rasburicase and Pegloticase
    • Special cases: Rasburicase used to prevent tumor lysis syndrome; Pegloticase for long-term gout management
  • Allopurinol and xanthine oxidase inhibition details
    • Allopurinol is metabolized to oxypurinol and inhibits XO; results in accumulation of hypoxanthine and xanthine, which can be salvaged/cleared by kidney
    • Febuxostat more effective in some patients; XO inhibition reduces uric acid formation
  • ADA deficiency (adenosine deaminase deficiency)
    • ADA converts adenosine/deoxyadenosine to inosine/deoxyinosine; highest activity in thymus and lymphocytes
    • ADA deficiency leads to accumulation of adenosine/deoxyadenosine→dATP, which inhibits ribonucleotide reductase, blocking DNA synthesis and causing SCID (T, B, NK cell deficiency)
    • Genetics: autosomal recessive; incidence ~1 in 200,000 births; accounts for ~1/3 of autosomal recessive SCID
  • Purine nucleoside phosphorylase (PNP) deficiency
    • Leads to impaired T-cell function with milder immunodeficiency than ADA deficiency

XI. Key clinical concepts and therapeutic implications

  • Folate (folic acid) and nucleotide metabolism
    • Folate derivatives (e.g., N^10-formyl-THF, N^5,N^10-methylene-THF) are essential cofactors in purine and pyrimidine synthesis; folate pathway inhibitors are widely used in chemotherapy (antimetabolites)
  • Antimetabolites and antibiotics implications
    • Many chemotherapeutic agents target folate metabolism or nucleotide synthesis; understanding salvage and de novo pathways helps explain drug actions and resistance
  • Real-world relevance
    • Gout management illustrates balance between production and excretion of uric acid and the importance of tailoring therapy to individual renal function and urate production status
    • Genetic enzyme deficiencies (HGPRT, ADA, PNP) reveal how disruptions in nucleotide metabolism can lead to profound immunological and metabolic disorders
  • Ethics and practice considerations
    • Genetic testing and counseling for hereditary metabolic disorders (e.g., LNS, ADA-SCID) are critical; therapy choices may involve enzyme replacement, gene therapy, or transplantation considerations
  • Connections to prior principles
    • Nucleotide metabolism integrates central pathways: energy (ATP), one-carbon metabolism (folate), amino acid metabolism, and redox (NADPH) balance
    • Regulation by feedback and feedforward mechanisms maintains nucleotide pools compatible with DNA replication and RNA transcription

XII. Key numerical values and formulas (summary)

  • SUA reference ranges:
    • Male: 3.4\text{-}7.0\;\text{mg/dL}
    • Female: 2.4\text{-}6.0\;\text{mg/dL}
  • Acute urate saturation threshold: SUA > 6.8\;\text{mg/dL}
  • Gout incidence from hyperuricemia: up to ~1/3 of individuals with hyperuricemia develop gout
  • ADA deficiency incidence: roughly 1/200{,}000 live births
  • Mechanistic notes (key reactions)
    • Purine base activation: ext{Ribose-5-phosphate} + ext{ATP} \rightarrow \text{PRPP} + \text{AMP}
    • GPAT regulation: PRPP activates; AMP/GMP inhibit
    • IMP as purine branching point to AMP or GMP
    • AMP synthesis: requires GTP and aspartate; GMP synthesis: requires ATP and glutamine
    • Pyrimidine synthesis: CPS II forms carbamoyl phosphate from glutamine and CO2; UTP inhibits CPS II; PRPP activates
    • UMP → UTP → CTP; thymidylate synthesis from dUMP via thymidylate synthase using N^5,N^10-methylene-THF
    • DNA precursor synthesis: NDPs reduced to dNDPs by ribonucleotide reductase using NADPH; dNTPs balanced by allosteric control
    • Purine catabolism: adenosine deaminase converts AMP to IMP; XO oxidizes hypoxanthine to uric acid
  • Linkage between folate metabolism and nucleotide synthesis underpins many cancer therapies (e.g., methotrexate inhibits folate cofactors)
  • Distinguish between acute gout management (reduce inflammation) and long-term control (lower SUA)
  • Recognize that not all hyperuricemia leads to gout; risk depends on urate crystal formation and inflammatory predisposition
  • Recall that salvage pathways are energy-efficient backups; defects cause shifts toward de novo synthesis or alternative metabolic stresses

Suggested Reading

  • Lippincott's Illustrated Review of Biochemistry, 8th edition (Abali, Cline, Franklin & Viselli), Chapter 22: 324–339

Study questions (from transcript)

  • de novo purine nucleotide synthesis takes place primarily in the liver; salvage occurs in all tissues; step 3–11 build IMP; AMP/GMP regulation; dNTP balance via ribonucleotide reductase; ADA deficiency leads to SCID; gout treatment choices include NSAIDs, Allopurinol, Febuxostat, Rasburicase, Probenecid; uric acid management depends on excretion and production profiles