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 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.

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

Numbering of sugar carbons with ‘prime’

Adenosine monophosphate Nomenclature Adenine BASE Adenosine NUCLEOSIDE Adenosine monophosphate (AMP) NUCLEOTIDE Another name for nucleotide is Adenylic Acid/Adenylate 5

Active forms of nucleotides: di-and tri-phosphates GMP + ATP GDP + ADP Nucleoside Monophosphate Kinase (i) To get active form of nucleotindes Specific reaction for cytidine MP General for generating the triphosphate XDP + YTP XTP + YDP Nucleoside Diphosphate Kinase (ii)

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

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

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.

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

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)

De novo biosynthesis of purines (A and G): activation of ribose-phosphate Ribose phosphate pyrophospho- Kinase (PRPP synthase) Pentose phosphate pathway IMP, AMP, GMP In unit 4, energy metabolism, Dr. Michael Lea discussed the Pentose Pathway

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

Purine biosynthesis intermediates Carboxylation unusual in not using biotin.

Purines: where do the atoms come from? Key: Glycine 1 C unit of N10 f-TetraHydroFolic acid (sulfonamides; methotrexate) Glutamine Asparate 17

Amino acids utilized in purine biosynthesis Carboxylation unusual in not using biotin. Inosine monophosphate is the precursor for both AMP and GMP

AMP & GMP synthesis IMP 19

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

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

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

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

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

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

Purines in humans are degraded to urate (ADA)

Gouty Arthritis The Gout By Royal Authority James Gilray 1799 Classification of Hyperuricemia Overproduction of urate • Primary idiopathic hyperuricemia • Hypoxanthine-guanine phosphoribosyl-transferase deficiency • Phosphoribosylpyrophosphate synthetase overactivity • Hemolytic processes • Lymphoproliferative disease • Myeloproliferative disease • Polycythemia vera • Psoriasis (severe) • Paget's disease • Rhabdomyolysis • Exercise • Alcohol • Obesity • Purine-rich diet Decreased excretion of uric acid • Primary idiopathic hyperuricemia • Renal insufficiency • Polycystic kidney disease • Diabetes insipidus • Hypertension • Acidosis --Lactic acidosis --Diabetic ketoacidosis • Down syndrome • Starvation ketosis • Berylliosis • Sarcoidosis • Lead intoxication • Hyperparathyroidism • Hypothyroidism • Toxemia of pregnancy • Bartter's syndrome Decreased excretion of uric acid (continued) • Drug ingestion --Salicylates (less than 2 g per day) --Diuretics --Alcohol --Levodopa-carbidopa (Sinemet) --Ethambutol (Myambutol) --Pyrazinamide --Nicotinic acid (niacin; Nicolar) --Cyclosporine (Sandimmune)   Combined mechanism • Glucose-6-phosphate dehydrogenase deficiency • Fructose-1-phosphate aldolase deficiency • Alcohol • Shock By Royal Authority ‘King George IV’ George Cruickshank 19th C.

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 Colchisine exerts its effect by inhibiting the phagocytosis of uric acid and blocking the release of chemotactic factor.

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

PRPP 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)

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

GOUT Treatment Guanine Xanthine Hypoxanthine Urate Allopurinol: xanthine oxidase People with high Uric acid levels may or may not have gout. But all people with Gout have NaUrate crystals in PMLs Allopurinol: a. decrease urate b. increase xanthine & hypoxanthine c. decrease PRPP

Initiate urate lowering therapy !! Tophi at helix of ear 1st MTP joint (75%) in first metatarsophalangeal joint (big toe: podagra), -tophi occur approximately 10 years after first attack -tophi seen in synovium, tendons, bursae and subcutaneous tissue -aspiration demonstrates uric acid crystals and inflammatory cells -incidence of tophi has been greatly decreased by the advent of medications to lower uric acid Synovial fluid: -monosodium urate crystals (intracellular)-needle-shaped crystals that are negatively birefringent under polarized light-diagnostic for gout -increased leukocytes: >15,000/cc WBCs, primarily PMNs C. Tophi aspirate: monosodium urate crystals-diagnostic Only 5% of persons with hyperuricemia develop gouty arthritis B. 7% of persons will have only one arthritic attack C. 60% will have recurrence within one year D. With prolonged disease, frequency of attacks increases and may even become chronic D. Renal dysfunction seen in 90% of persons with gouty arthritis however, severe renal dysfunction is uncommon. Renal dysfunction may not be due to hyperuricemia, but rather may be due to other concomitant diseases, e.g. HTN, diabetes normal: males 3-8mg/dl females 1.5-6mg/dl ("normal" values may vary between laboratories) Large tophaceous deposits surrounding joint Initiate urate lowering therapy !!

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. alcohol metabolism produces net adenosine triphosphate degradation to adenosine monophosphate, which is subsequently converted to uric acid. In addition, lactate generated via alcohol consumption increases proximal tubular urate re-absorption while interfering with urinary urate secretion.[36,37] Chronic heavy use of alcohol also has the potential to inhibit conversion of the pro-drug allopurinol to its active metabolite oxypurinol.[38] Beer contains purines

Classification of Hyperuricemia Overproduction of urate • Primary idiopathic hyperuricemia • Hypoxanthine-guanine phosphoribosyl-transferase deficiency • Phosphoribosylpyrophosphate synthetase overactivity • Hemolytic processes • Lymphoproliferative disease • Myeloproliferative disease • Polycythemia vera • Psoriasis (severe) • Paget's disease • Rhabdomyolysis • Exercise • Alcohol • Obesity • Purine-rich diet Decreased excretion of uric acid • Primary idiopathic hyperuricemia • Renal insufficiency • Polycystic kidney disease • Diabetes insipidus • Hypertension • Acidosis --Lactic acidosis --Diabetic ketoacidosis • Down syndrome • Starvation ketosis • Berylliosis • Sarcoidosis • Lead intoxication • Hyperparathyroidism • Hypothyroidism • Toxemia of pregnancy • Bartter's syndrome Decreased excretion of uric acid (continued) • Drug ingestion --Salicylates (less than 2 g per day) --Diuretics --Alcohol --Levodopa-carbidopa (Sinemet) --Ethambutol (Myambutol) --Pyrazinamide --Nicotinic acid (niacin; Nicolar) --Cyclosporine (Sandimmune)   Combined mechanism • Glucose-6-phosphate dehydrogenase deficiency • Fructose-1-phosphate aldolase deficiency • Alcohol • Shock 35

Lesch-Nyhan Syndrome Severe HGPRT deficiency: decreased IMP&GMP 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 36

Severe Combined Immunodeficiency Syndrome 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 AMP Adenosine Inosine H20 Pi NH3 Hypoxanthine Nucleotidase Adenosine deaminase* infusion of autologous CD34+ cells from bone marrow that had been transduced with a viral vector carrying the ADA gene.; 10 out of ten children doing well after mean survivial of 4 yrs (1.8-8).

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

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

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

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