METABOLISM OF PURINE NUCLEOTIDES & PURINE DISORDERS

1. METABOLISM OF PURINE NUCLEOTIDES & PURINE DISORDERS

2. ROLES OF NUCLEOTIDES • Building blocks for DNA and RNA • “Second messengers” in signal transduction cascades • Energy “currency” of the cell • Components of major co-enzymes

3. Purine Biosynthesis • Major site – (Adenine & Guanine) in the Liver. • Synthesis starting from Ribose-5-phosphate Denova pathway:Precursors contribute to form the Purine ring: Glycine(C4, C5, N7),Glutamine( N3, N9),THF(C2, C8 atoms)Aspartate(N1 atom), & CO2(C6 atom). • The construction of 6-membered ring forms Inosine- 5-monophosphate (IMP) – 1st Purine. • Energetics – expenditure of 6 high energy bonds. Salvage pathway- Free & dietary purine can be converted into corresponding nucleotides (no need ATP). • RBC, Neutrophils & Braincannot produce Purines by de novo synthesis. But by only salvage pathway (HGPRTase).

4. ATP AMP 5-phosphoribosylpyrophosphate (PRPP) PRPP synthetase Gln PRPP amidotransferase Amino acids: Gly + Gln + Asp Glu Inosine mono phosphate (IMP) 5-phospho-ribosylamine Cofactors: N10-formyl THF • Glucose-6-P pentose phosphate pathway Ribose-5-phosphate Figure 1.Synthesis of inosine monophosphate (IMP)

5. NAD+ aspartate + GTP GDP + Pi NADH Gln + ATP fumarate Glu+AMP+PPi guanosine monophosphate (GMP) adenosine monophosphate (AMP) GDP ADP GTP ATP IMP Figure 2.Formation of AMP and GMP from IMP

6. Ribose-5-phosphate allosteric inhibition PRPP synthetase PRPP PRPP amido-transferase 5-phospho-ribosylamine IMP GMP AMP GDP ADP Figure 3.Allosteric inhibition of purine biosynthesis; also ATP stimulates formation of AMP. Regulatory enzyme is PRPP amido transferase, controlled by feed back inhibition of nucleotides - IMP, AMP & GMP.

7. +PRPP +PRPP HGPRT xanthine oxidase HGPRT Xanthine xanthine oxidase IMP GMP Uric acid GMP, GDP or GTP AMP, ADP or ATP Adenosine Guanosine NH3 adenosine deaminase Inosine Ribose-1-P purine nucleoside phosphorylase Ribose-1-P Guanine Hypoxanthine Figure 4. Degradation of purines to uric acid and salvage of purine basesvia hypoxanthine-guanine phosphoribosyl transferase

8. METABOLISM OF PURINE

9. ATP AMP 5-phosphoribosylpyrophosphate (PRPP) PRPP synthetase Gln PRPP amidotransferase Amino acids: Gly + Gln + Asp Glu Inosine mono phosphate (IMP) 5-phospho--ribosylamine Cofactors: N10-formyl THF • Glucose-6-P pentose phosphate pathway X1a Ribose-5-phosphate Figure 1.Hyperuricemia: Gout: X1a = PRPP synthetase defects associated with a superactive enzyme characterized by an increased Vmax or an enzyme with a reduced Km for ribose-5-P.

10. Disorder of Purine catabolism • In humans, Purine rings are degraded to the metabolically inert uric acid. • Normal level of uric acid in blood is 2-7 mg/dl (Female) & 3-8 mg/dl (Male). • Uric acid excretion in urine is about 500 – 700 mg/day. 1. GOUT (Hyper uricemia) • Aninherited metabolic disease • Incidence - 3 /1000 person • Defect - partial deficiency of HGPRTase or increased levels of PRPP caused by a hyperactive synthetase. • Causes - ↓ Synthesis of GMP and IMP & ↑ in PRPP levels --> ↑d Purine biosynthesis by de novo pathway.

11. Gout 1. Primary gout: • Inborn error of metabolism due to over production of uric acid • Causes:Varient PRPP synthetase, PRPP gltamyl amido transferase, HGPRT deficiency, Glu-6-Pase defiecincy. 2. Secondary gout: • Various disease causing ↑or ↓uric acid excretion. • E.g.: Cancer, Psoriasis, Trauma, Starvation, Impairment in renal function leads to gout.

12. GOUT Causes • Uricosuria • Accumulation of sodium urate crystals in the soft tissues called Tophi -causes painful Gouty - Arthritis. • Deposition of Na-urate crystals -Renal calculi • Clinical finding- Red faced, Acidosis & Renal damage. Treatment • Reduced dietary Purine intake & restrict alcohol is advised. • Uricosuric drugs. For example – Probenecid (Benemide), Salicylates and Halofenate. • Allopurinol (analog of hypoxanthine) competitively inhibits Xanthine oxidase & thus uric acid synthesis (Suicide inhibition) • Coichicineis an anti-inflammatory drug to relieve pain. Other useful drugs include Indomethacin, Naproxen, Brufen, Corticosteroids etc.

13. 2. LESCH-NYHAN SYNDROME • X-linked recessive disorder of Purine metabolism. • Incidence is 1 in 10,000 males • Defect- Total lack of HGPRTase, salvage pathway • Thus ↑ PRPP (de novo pathway), ↑ production of uric acid. • Diagnosis - Murexide test (reddish deposit) to detect urine uric acid Clinical manifestations • ↑ Production of uric acid causes severe gout, poor growth, and renal failure due to Nephrolithiasis. • Neurological abnormalities such as Mental retardation, Aggressive behavior, learning disability & compulsive self-destructive behavior. • Irresistible urge to bite their finger & lips (Self mutilation) • It shows that abnormal behavior can be caused by absence of a single enzyme.

14. GMP, GDP or GTP AMP, ADP or ATP Adenosine Guanosine NH3 Adenosine deaminase Inosine Purine nucleoside phosphorylase Ribose-1-P Ribose-1-P +PRPP Guanine Hypoxanthine +PRPP HGPRTase xanthine oxidase HGPRTase Xanthine xanthine oxidase IMP GMP Uric acid X2 X2 Inhibited by allopurinol Figure 4. X2 = moderate defect (>50% activity) leading to gout; severe defect (very low activity) leads to Lesch-Nyhan syndrome.

15. 3. Immunodeficiency diseases • Immunodeficiency diseases are associated with Purine degradation disorders. • Defect: Adenosine deaminase & Purine nucleoside phosphorylase involved in uric acid synthesis. • Causes: Severe combined immunodeficiency (SCID) involving T-cell & usually B-cell dysfunctions and thus impaired the immunity. • Uric acid synthesis ↓& tissue level of N.S & N.T↑ • Treatment: Transferring ADA gene by Gene therapy.

16. 4. Hypo uricemia Hypoxanthine xanthine oxidase • When serum uric acid level is less than 2 mg/dl represents hypouricemia. • A rare congenital Xanthine oxidase deficiency • Incidence is 1 in 45,000. • It leads to the increased excretion of Xanthine & Hypoxanthine. • Causes: Excretion of xanthine in urine - Xanthinuria. Frequently causes the formation of Xanthine stones in the urinary tract. Xanthine xanthine oxidase Uric acid

17. SYNTHESIS OF PYRIMIDINE NUCLEOTIDES & PYRIMIDINE DISORDERS

18. SYNTHESIS OF PYRIMIDINE NUCLEOTIDES • Synthesis of the Pyrimidine is less complex than that of the purines, since the base is much simpler (6 member ring). • Precursors –Carbomyl phosphate & Aspartate • De novo synthesis - in the Liver cytosol • Energetic: Requires 2ATP

19. Carbamoyl phosphate synthetase II (gln) Aspartate transcarbamoylase Carbamoyl-aspartate Glutamine + 2ATP + CO2 Aspartate Glu + Pi + 2 ADP Orotate Glu + Pi + ADP Gln + ATP +PRPP Orotate phosphoribosyl transferase +2 ADP +2 ATP UTP UMP OMP CTP Orotidylic acid decarboxylase  ATP, PRPP Carbamoyl phosphate - UDP UTP Figure1 -Biosynthesis of the Pyrimidine nucleotides UTP & CTP

20. CPS-II Asp. Trans carbomylase Regulation of Pyrimidine Biosynthesis OMP Decarboxylase

21. CATABOLISM OF PYRIMIDINE NUCLEOTIDES • Pyrimidine degraded in the liver. • The end products - are nitrogenous bases Cytosine, Uracil and Thymine. • The bases are then degraded to amino acids, namely β-Alanine (from Cytosine & Uracil) & β-Amino isobutyrate(from Thymine). • These amino acids undergo transamination & other reactions to finally produce Acetyl coA, Succinate & CO2. • Clinical significance: β-Aminoisobutyrate is excreted in large quantity in Leukemia & when body subjected to X-ray irradiation.

22. 2. Reye’s syndrome • Secondary Oratic aciduria • Defect in Ornithine transcarbamoylase (in urea cycle) Causes • Accumulation of Carbomyl phosphate • This is then diverted for the increased synthesis and excretion of oratic acid.

23. Clinical significances of pyrimidine metabolism Oraticaciduria • Rare inherited disorders. • Enzyme deficiency- Orotate phosphoribosyl transferase and OMP decarboxylase. • Defect - in UMP formation & thus ↑ Oratate synthesis in blood & its excretion in the urine. • Causes - Growth retardation, severe anemia caused by hypo chromic erythrocytes and megaloblastic bone marrow. Leukopenia is also common. • Treatment – Diet rich in uridine & cytidine, which ↑UMP production via the action of nucleoside kinases. UMP then inhibits CPS-II, thus ↓ orotic acid production.