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Lecture 29 Purine Metabolism/Diseases

Lecture 29 Purine Metabolism/Diseases. Raymond B. Birge, PhD. Nucleotides - key roles in cellular processes:. 1. Activated precursors of RNA and DNA 2. Adenine nucleotides are components of the major co-enzymes, NAD, NADP, FMN, FAD, and CoA

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Lecture 29 Purine Metabolism/Diseases

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  1. Lecture 29 Purine Metabolism/Diseases Raymond B. Birge, PhD

  2. Nucleotides - key roles in cellular processes: 1. Activated precursors of RNA and DNA 2. Adenine nucleotides are components of the major co-enzymes, NAD, NADP, FMN, FAD, and CoA 3. Nucleotide derivatives are activated intermediates in biosynthetic processes (UDP-glucose, SAM) 4. Serve as metabolic regulators (e.g cAMP and the activation of cell signaling). 5. Serve as major currency of energy in all cells (ATP and GTP). 6. Several metabolic diseases have their etiology in nucleotide metabolism.

  3. Purine metabolism (Overview) 1. Nomenclature/nucleotide structure 2. De novo synthesis pathways 3. Re-utilization (salvage) pathways 4. Degradation pathways 5. Metabolic diseases of purine metabolism (Gout, Lesch-Nyhan, SCID) Suggested reading: Lippencott’s Chapter 22

  4. Adenine Adenosine monophosphate (AMP) Adenosine NUCLEOTIDE NUCLEOSIDE BASE Nomenclature

  5. Active forms of nucleotides: di-and tri-phosphates (i) Nucleoside Monophosphate Kinase GMP + ATP GDP + ADP (ii) Nucleoside Diphosphate Kinase XDP + YTP XTP + YDP

  6. Why are nucleosides and nucleotides important For biochemists? Purine binding proteins (“the purine proteome”) comprise a family of 3-4,000 Proteins and as much as 50% of all druggable targets in biology. Kinases Helicases Reductases Transferases Synthetases Dehydrogenases Chaperones Metabolic Enzymes DNA and RNA processing Etc

  7. Common Purine Bases NH2 O Adenine Hypoxanthine O O O NH2 Xanthine Guanine Guanine H= 6 oxy purine X= 2,6 dioxy purine A= 6 amino purine G= 2 amino, 6-oxy purine

  8. Nucleoside Function in extracellular signal transduction Adenosine nucleoside-increased during ATP degradation. Released in cells when there is low O2 concentration Binds to purinogenic receptors A1, A2A, A2B, A3 Slows the heart down, at the same time increases capillary dilation Caffeine is a adenine derivative, and antagonizes the effects of adenine.

  9. 10-10 M cAMP PKA P P P phosphorylase kinase glycogen synthase ATP phosphorylase Cyclic nucleotides are important mediators for Intracellular signal transduction

  10. Two pathways to generate purines and pyrimidines 1. DE NOVO BIOSYNTHETIC PATHWAYS (building the bases from non-purine molecules) 2. SALVAGE PATHWAYS (the reutilization of bases from dietary or catabolic sources)

  11. De novo biosynthesis of purines (A and G): activation of ribose-phosphate Pentose phosphate pathway Ribose phosphate pyrophospho- Kinase (PRPP synthase) IMP, AMP, GMP

  12. Major regulatory step in purine biosynthesis: PRPP to 5-Phosphoribosyl-1-amine * Glutamate Glutamine PPi PRPP Amidophosphoribosyl transferase Inhibited by products IMP, AMP, and GMP. FEEDBACK INHIBITION

  13. Purine biosynthesis intermediates

  14. Purines: where do the atoms come from? Key: Glycine 1 C unit of N10 f-TetraHydroFolic acid (sulfonamides; methotrexate) Glutamine Asparate

  15. Amino acids utilized in purine biosynthesis Inosine monophosphate is the precursor for both AMP and GMP

  16. AMP & GMP synthesis IMP

  17. Hypoxanthine to Adenine/Guanine. O NH2 (N source) Aspartate Hypoxanthine-IMP Adenine O O (N source) Glutamine O NH2 Guanine Xanthine-XMP The common mechanistic theme for the conversion to A and G is the conversion of a carbonyl oxygen to an amino group

  18. Ribose 5-phosphate PRPP Regulation of purine de novo biosynthesis: classic negative feedback Phosphoribosyl amine Inhibited by AMP Inhibited by IMP, AMP, and GMP AMP IMP GMP Inhibited by GMP

  19. O A O P O CH2 O PPi + Base (Adenine or Guanine) + OH OH OH O O P O CH2 PPi O OH OH OH Adenylate/AMP PRPP + Adenine Salvage pathways: re-utilizate purines There are 2 salvage enzymes with different specificities: 1. Adenine phosphoribosyl transferase (APRT) 2. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) A-PRT HG-PRT Guanylate/GMP PRPP + Guanine

  20. Endonuclease Nucleic Acid Synthesis Nucleoside triphosphate Phosphodiesterase Nucleoside monophosphates (Mononucleosides) Stages of nucleotide metabolism

  21. Nucleoside monophosphates (Mononucleosides) Endonuclease Nucleic Acid Synthesis Nucleoside triphosphate Phosphodiesterase H20 Nucleotidases PPi Pi ADP Phosphoribosyl transferases Nucleoside kinase Nucleosides ATP Pi PRPP Phosphorylases Ribose-1-P Nucleobases (A, G) Uric Acid (purines)

  22. Getting Back to ‘Basics’ Nucleotidase Adenine Phosphorylase Adenosine monophosphate (AMP) Adenosine NUCLEOTIDE NUCLEOSIDE BASE

  23. Purines in humans are degraded to urate (ADA)

  24. Gouty Arthritis The Gout James Gilray 1799 By Royal Authority ‘King George IV’ George Cruickshank 19th C.

  25. Some famous people who had gout Henry VIII Kublai Khan Nostradamus John Milton Isaac Newton Frederick the Great John Hancock Thomas Jefferson Benjamin Franklin David Wells

  26. Gout results from HYPERURICEMIA Decreased URIC ACID excretion: 80% of gout - ideopathic, renal disease, diabetes insipidus, hypertension, Downs syndrome, many others Increased URIC ACID production: 20% of gout – PRPP synthase overactivity, hemolytic diseases, lymphoproliferative diseases, may others HGPRT deficiency (Lesch Nyhan Syndrome), exacerbated by alcohol, purine rich diet, obesity

  27. Gout from increased uric acid production 1. Lost regulation of PRPP Synthase & PRPP Amidotransferase Ribose 5-phosphate Phosphoribosyl amine PRPP purines Inhibited by IMP, AMP, and GMP Leads to net increase in biosynthetic/degradation pathways!! (From slide #18)

  28. Gout from increased uric acid production 2. Defects in salvage pathway lead to increased PRPP & Guanine [HG-PRT] GMP PRPP + Guanine Leads to net increase in biosynthetic/degradation pathways!!

  29. GOUT Treatment Hypoxanthine Guanine xanthine oxidase Xanthine Urate xanthine oxidase NaUrate crystals in PMLs Allopurinol: a. decrease urate b. increase xanthine & hypoxanthine c. decrease PRPP

  30. Tophi at helix of ear 1st MTP joint Large tophaceous deposits surrounding joint Initiate urate lowering therapy !!

  31. Clinical Significance of Purine metabolism ID: A 56 year old obese white male comes to his family doctor. Chief Complaint: ‘My big toe hurts like !*?!#*!?!!!’ History Present Illness: Pain began during the night after an episode of binge eating and drinking. Past Medical History: Significant for removal of kidney stones last year. Current Health/Risk Factors: He admits to being an avid meat eater and drinks beer every night. Physical Exam: fever, right metatarsophalangial (MTP) joint red, hot and swollen; painful to motion; subcutaneous deposits in helix of left ear Pathology: Synovial fluid aspiration shows negatively birefringent, needle-shaped crystals.

  32. Lesch-Nyhan Syndrome X-linked recessive Severe HGPRT deficiency: decreased IMP&GMP increased PRPP & de novo purine p’way Hyperuremia: gouty arthritis, kidney stones, tophi Neurologic disability: spasticity, hyperreflexia Behavioral problems: cognitive dysfunction, aggression, self-injury

  33. AMP H20 Nucleotidase Pi Adenosine H20 Adenosine deaminase* NH3 Hypoxanthine Inosine SCID Severe Combined Immunodeficiency Syndrome Autosomal recessive disorder Mutations in ADA* Infants subject to bacterial, candidiasis, viral, protazoal infections Both T and B cells reduced (dATP is toxic) 1995-AdV expressing ADA was successfully employed as gene therapy strategy

  34. Adenosine Deaminase (ADA) deficiency Urate (From slide #19)

  35. Bottom Line Recognize names and structures of purines/nomenclature of NMPs: Adenosine, Guanine, Hypoxanthine, Xanthine Name the precursors of atoms in the purine ring: Gln (N); Gly (C, N); N10-fTHF (C ); HCO3-(CO); Asp (N) Recognize the regulated reactions: PRPP synthase: IMP, AMP, GMP Gln:PRPP amidotransferase: IMP, AMP, GMP; PRPP IMP AMP: AMP IMP GMP: GMP Explain the cause of SCIDS Make differential diagnosis of gouty arthritis and Lesch-Nyhan Syndrome Explain mechanisms of the following treatments: sulfaonamides, methotrexate, allopurinol

  36. Summary and Take-Home Points • Identify basic structures of purines, nucleosides, and • nucleotides. • 2. Identify key relationships between glucose metabolism • and purine biosynthesis. • 3. Knowledge of how amino acids are used in AMP and • GMP biosynthesis. • 4. Understand degradation pathways of purines and • their relationship to uric acid metabolism and gout

  37. Integrative Thought Question Ribonucleotides and Deoxyribonucleotides are essential for all cells, and represent key convergent points in energy metabolism. Provide specific examples in which purine metabolism can be linked to metabolism of 1). Glucose 2). Lipids 3). Amino Acids 4). Ammonium

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