NUCLEOTIDE METABOLISM Metabolism of purine nucleotides Prof. Mária Sasvári NUCLEOTIDE METABOLISM Metabolism of purine nucleotides Gergely Keszler 2009.

The biological role of nucleotides Building blocks of nucleic acids (DNA and RNA) Storage of biochemical energy (ATP and GTP) Activation of biosynthetic precursors (UDP-glucose, CDP-choline) Components of coenzymes (NAD, FAD, Coenzyme A etc.) Regulation of metabolism (cAMP, cGMP) 6. Nucleotide analogues: anticancer and antiviral therapies

Terminology of nucleotides phosphoanhydride bonds phosphoester bond N-glycosidic bond Nucleotides are composed of: a nucleobase a pentose at least one phosphate group a nucleoside

Ribo- and deoxyribonucleotides (Ado) (dAdo) (Guo) (Urd) (Cyd) (dGuo) (Thd) (dCyd)

Structures of nucleobases N-containing, heterocyclic aromatic compounds; substituted purine or pyrimidine rings RNA DNA

Nucleotide synthesis „de novo” salvage (recycling) stepwise assembly from small precursors (C1 fragments, CO2, amino acids, ribose-P) salvage (recycling) • base + ribose-P → nucleotide (typical for purines) OR • nucleoside + Pi → nucleotide (typical for pyrimidines)

“de novo” and salvage reactions The origin of ribose-P glc-6-P fru-6-P PPP O-P P 1’ P-O-H2C 5’ O ATP AMP ri-5-P PRPP synthetase PRPP Pyrimidine “de novo” synthesis Purine “de novo” and salvage reactions

 “de novo” synthesis URINE Intestine Blood brain, RBC, lymphocytes Food  RNA, DNA polynucleotides nucleotides nucleosides bases “salvage reactions” “de novo” synthesis  nucleotides nucleosides bases  urate nucleosides bases liver DNA RNA urate  URINE

Purine nucleotide synthesis ATP  ADP AMP GTP  GDP GMP IMP salvage reactions purine bases “de novo” synthesis

The origin of the purine ring IMP CO2  Asp 2 5 N Glycine N 6 7 3 4 1 N N N10formyl H4F N10formyl H4F Gln

“de novo” purine synthesis (5-phosphoribosyl-1-amine) Gln N 10 formyl H 4 F Glycine CO 2 ¯ Asp “de novo” purine synthesis PRPP 1. Gln + H2O Gln PRPP amidotransferase Glu + PPi NH3+ 9. ri-5-P PRA (5-phosphoribosyl-1-amine) Glycine 2. ATP ADP + Pi GAR synthetase

“de novo” purine synthesis NH NH3+ 9. O ri-5-P GAR (5’PR-Glycinamide) 3. N10formyl H4F H4F GAR formyltransferase

“de novo” purine synthesis (5’PR-formyl-glycinamide) NH O ri-5-P O FGAR (5’PR-formyl-glycinamide) 4. Gln Glu ATP ADP + Pi FGAM synthetase

“de novo” purine synthesis (5’PR-formylglycinamidine) NH O ri-5-P H2N HN FGAM (5’PR-formylglycinamidine) 5. ATP ADP + Pi AIR synthetase

“de novo” purine synthesis (5’PR-5-amino-imidazole) ri-5-P H2N AIR (5’PR-5-amino-imidazole) 6. CO2 AIR carboxylase

“de novo” purine synthesis (5’PR-4-Carboxy- 5-amino-imidazole) -OOC N H2N ri-5-P CAIR (5’PR-4-Carboxy- 5-amino-imidazole) 7. Asp ATP ADP + Pi SAICAR synthetase

“de novo” purine synthesis H2N ri-5-P O HN succinyl- SAICAR (5’PR-succinyl-5-aminoimidazole-4-carboxamide) 8. Adenylosuccinase (ASA) fumarate

“de novo” purine synthesis (5’PR-5-aminoimidazole-4-carboxamide H2N ri-5-P O H2N ACAIR (5’PR-5-aminoimidazole-4-carboxamide 9. N10formyl H4F H4F AICAR transformylase

“de novo” purine synthesis 5’PR-5-formamidoimidazole-4-carboxamide) ri-5-P O H2N FACAIR 5’PR-5-formamidoimidazole-4-carboxamide) 10. IMP cyclohydrolase H2O IMP

“de novo” purine synthesis and the purine nucleotide cycle AMP (6-amino) GMP (2-amino-6-oxo) AMP DA AMP+PPi ASL GS fumarate ATP Gln Xanthylate (2,6,-dioxo) Adenylosuccinate ASS GTP GDP+Pi NAD+ H2O NADH + H+ IMPDH Asp IMP(6-oxo)

The role of the purine nucleotide cycle Substrate level/oxidative phosphorylation - ADP + Pi  ATP + AMP 2 ADP ATP AMP kinase PNC AMP DA IMP AMP urate Adenylosuccinate severe Pi deficiency  [AMP]     hyperuricaemia e.g. fructose intolerance

Muscle: high AMP DA level Liver ATP AMP IMP NH3 + glycolysis NH3 inosine  urate inosine urate strenuous exercise: NH3 , urate  Muscle AMP DA def.: cramps, NH3, urate is NOT elevated

“de novo” purine synthesis Summary No free purine base during synthesis Carbon donors: „C1 units” (N10-formyl-THF) CO2 Glycine N-donors: Asp Gln Gly Energy: 6 ATP for 1 IMP Multifunctional proteins

Regulation of de novo purine synthesis + + “salvage” - - Gln PRPP amidotransferase + PRPP - IMP, GMP, AMP PRPP synthetase - ATP,GTP

Purine salvage reactions PRT (phosphorybosyl transferase) base nucleotide PRT (phosphorybosyl transferase) + ribose-P APRT adenine AMP PRPP PPi HGPRT hypoxantine guanine PRPP PPi IMP GMP

The Lesch-Nyhan syndrome linked to X-chromosome HGPRT deficiency: low GTP levels in the basal ganglia Hyp/G + PRPP → IMP/GMP + PPi linked to X-chromosome mental retardation self-mutilation aggression hyperuricemia

Catabolism of purine nucleotides PNP (purine nucleoside AMP GMP Pi 5’nucleotidase B r adenosine (6-amino) guanosine H2O NH3 ADA adenosine deaminase ri-1-P Pi inosine (6-oxo) PNP (purine nucleoside phosphorylase) B hypoxantine guanine

(2-oxo-6-amino-purine) Purine salvage reactions hypoxantine (6-oxo-purine) guanine (2-oxo-6-amino-purine) E x c r e t i o n H2O NH3 guanase H2O + O2 H2O2 xanthine (2,6-dioxopurine) xanthine oxidase urate (2,6,8-trioxopurine) URINE H2O + O2 H2O2

urate (dissociated anion) Why is uric acid acidic? uric acid (oxo) uric acid (enol) urate (dissociated anion) well soluble poor solubility precipitates in joints, initiates chemical arthritis GOUT

Hyperuricemia (gout) Symptoms: acute gouty arthritis urate crystals on the napkin (Lesch-Nyhan) Na-urate crystals  kidney stones urate in connective tissues and joints: „tophus”, inflammation, pain acute gouty arthritis chronic gouty arthritis Reason: Urate has low solubility (especially at acidic pH )

Reasons for hyperuricemia 1. PRPP overproduction as a consequence of mutation at the allosteric site of PRPP synthase, the enzyme cannot be inhibited overproduction of ribose-5-P PPP ri -5-P PRPP gl -6-P fr e.g. gl-6-phosphatase deficiency (von Gierke’s disease) Gl- 6-P   fr- 6-P   ri- 5-P 

Reasons for hyperuricemia Decreased adenine, guanine reutilization 2. Absence of purine salvage reactions e.g. HPRT deficiency Decreased adenine, guanine reutilization  increased excretion

Reasons for hyperuricemia 3. Low ATP level, disturbed ATP metabolism strenuous exercise fructose intolerance (phosphate trap) see before

Reasons for hyperuricemia overproduction of purines 4. Secondary reasons: tissue damage cancer, cell damage Overproduction of organic anions (lactate, ketone bodies, drug derivatives)  DNA breakdown overproduction of purines

Medication of gout: allopurinol hypoxanthine Xanthine oxidase xanthine allopurinol alloxanthine oxopurinol Competitive inhibitors Hypoxanthine and xanthine in urine (better solubility)

Allopurinol, a special purine analogue N-7 and C-8 have been scrambled up Blocks xanthine oxidase, the enzyme catalyzing the oxidation of xanthine to uric acid – cures gout

Enzyme deficiency: ADA / PNP / (ADA + PNP) Symptoms: immunodeficiency, “NON-HIV AIDS” Reason: B/T lymphocyte deficiency Mechanism: adenosine   dATP (ATP)  dATP  inhibits ribonucleotide reductase  inhibits DNA synthesis  promotes apoptosis Treatment: ADA enzyme therapy, gene therapy

ADA ADA Adenosine deaminase functions on the outer surface of red and white blood cell membranes (ectoenzyme) ADA ADA binding glycoprotein („complexing factor”)

A1 A2 The pathogenesis of SCID - selective lymphotoxicity Gi Gs 1. Extracellular accumulation of (deoxy)adenosine (d)Ado A1 A2 Gi Gs dAdo cAMP  cAMP  Inhibition of the SAM/SAH cycle Impaired DNA synthesis

The pathogenesis of SCID - selective lymphotoxicity apoptosis 2. Intracellular accumulation of (deoxy)adenosine triphosphate Inhibition of ribonucleotide reductase Inhibition of cell proliferation dCK dATP  dAdo dAdo dAMP AK DNA strand breaks Inhibition of DNA polymerases Lymphocyte- selectivity!! apoptosis

Clinical manifestation of SCID  Recurring, opportunistic infections: candidiasis, Pneumocystis-pneumonia  Absence of lymph nodes, no thymic shadow upon chest X-ray examination  Severe impairment of both humoral and cellular immunity:  lower than 500/μl total lymphocyte count  very low plasma immunoglobulin levels  Untreated patients die before their age of 2 years

Treatment of ADA deficiency 1. Treatment of symptoms  Infections antibiotics, antiviral and antifungal drugs  Immunoglobulin supplementation Maternal immunoglobulins are effective in the first few weeks of life

Treatment of ADA deficiency 2. Enzyme substitution  Red blood cell transfusion  Polyethylene-glycol-conjugated recombinanat ADA (PEG-ADA): intramuscular injection costs: 250,000 USD a year

Treatment of ADA deficiency 3. Gene therapy Principle: Introduction of the normal allele into the patients’ own stem cells Ex vivo: Stem cells are transfected and trans- planted into the patient

Ex vivo gene therapy

Ashanti da Silva: the first patient in the world treated by retrovirus-mediated ADA gene therapy The introduced ADA gene functioned fine for a few months. Later on, PEG-ADA substitution therapy must have been restarted due to inactivation of the gene.