1. DR. Saidunnisa M.D Associate Professor Department of Biochemistry Metabolism of Purines-Lecture

2. Learning Objectives  At the end of the session the student shall be able to Explain,enumerate, and define: 1. Enumerate the sources of individual atoms in purine ring. 2. Explain the major steps in the synthesis (denovo and salvage) and catabolism of purine. 3. Explain the Regulation. 4. Interpret disorders like Lesch-Nyhan syndrome, SCID, Adenosine deaminase deficiency and Gout.

3. Overview  Summary: 1. De novo synthesis of purines 2. Purine catabolism 3. Salvage pathway  Disorders: 1. Gout 2. SCID, (Adenosine deaminase deficiency) 3. Lesch-Nyhan syndrome

4. Nucleic acids  There are two types of nucleic acids namely DNA (Deoxy ribonucleic acid) and RNA (Ribonucleic acid) primarily they serve as transmitters of genetic information.  Nucleic acids are the polymers of nucleotides.

5. Bases  Purines and pyrimidines are major bases.  Purines are Adenine and Guanine.  Pyrimidines are Cytosine Uracil and Thymine

6. Bases-Rings  These are planar aromatic heterocyclic compounds.

7. PurinesPyrimidines

8. Sugars

9. Structure of nucleotides A phosphate group Nucleotides have three characteristic components : A nitrogenous base (pyrimidines or purine) A pentose sugar

10. Structure of nucleosides Remove the phosphate group, and you have a nucleoside. H

11. Digestion of Nucleic acids  Nucleic acids in the diet are hydrolyzed to a mixture of nucleotides by ribo and deoxy ribonuclease present in pancreatic and intestinal secretions.  Nucleotidases liberate phosphate from nucleotides resulting nucleosides are hdrolysed by nucleosidases forming free bases and pentose sugars.

12. Purines

13. Sources of carbon and nitrogen atoms in the purine ring  Purine ring is synthesized from different small components. Purines: where do the atoms come from?

14. De novo synthesis of Purines  These pathways use amino acids as precursors.  Liver is the main site of synthesis.  Operates in cytoplasm.  Starting material is ribose 5-phosphate produced in the HMP pathway.  The Purines are built upon a pre-existing ribose 5-phosphate.  Through a series of reactions, add the donated carbons and nitrogen's.

15. The biosynthesis of purine (A and G) begins with the synthesis of the ribose-phosphate The biosynthesis of purine (A and G) begins with the synthesis of the ribose-phosphate PRPP Synthase Pentose phosphate pathway Pentose phosphate pathway Preparatory step: Ribose-5phosphate, reacts with ATP to form phosphoribosyl pyrophosphate (PRPP) in the presence of PRPP Synthase. PRPP common substrate for synthesis of purines and pyrimidines.

16. Glutamine transfers its amide nitrogen to PRPP to replace pyrophosphate and produce 5-phospho ribosylamine. The enzyme PRPP Glutamyl amido transferase. This reaction is the “committed step” or “rate regulating step”. Glutamine transfers its amide nitrogen to PRPP to replace pyrophosphate and produce 5-phospho ribosylamine. The enzyme PRPP Glutamyl amido transferase. This reaction is the “committed step” or “rate regulating step”. PRPP Glutamine Glutamate PPi PRPP Glutamyl amido transferase * PRPP Glutamyl amido transferase is an important regulatory enzyme in purine biosynthesis. It is strongly inhibited by the end products IMP, AMP, and GMP. This type of inhibition is called FEEDBACK INHIBITION. PRPP Glutamyl amido transferase is an important regulatory enzyme in purine biosynthesis. It is strongly inhibited by the end products IMP, AMP, and GMP. This type of inhibition is called FEEDBACK INHIBITION. Step-1

17. Several amino acids are utilized in purine biosynthesis Mechanism of all the reactions are not required Step 3: Carbon 8 is: N10 methenyl THF4 Step-4: Nitrogen 3: Glutamine Step 5: Carbon 6 : CO2 Step 6: Nitrogen 1:Asparatate Step 7: Carbon 2 : THF4 Subsequent steps

18. Purine nucleotide synthesized is IMP is the precursor for both AMP and GMP

19. Ribose 5-phosphate PRPP Phosphoribosyl amine IMP AMP GMP Inhibited by IMP, AMP, and GMP Inhibited by AMP Inhibited by GMP The regulation of purine biosynthesis is a classic example of negative feedback The regulation of purine biosynthesis is a classic example of negative feedback PRPP Synthase PRPP Glutamyl amido transferase Intracellular concentration of PRPP regulates purine synthesis this in turn is dependent on the availability of ribose-5- phosphate and the enzyme PRPP Synthase.

20. Formation of purine nucleoside di and tri phosphates:  The nucleoside mono phosphates have to be converted to the corresponding di and tri phosphates to participate in the metabolic reactions.  This is achieved by the transfer of phosphate group from ATP, catalyzed by Nucleoside monophosphate and diphosphate kinases.

21. Conversion of Ribo to Deoxy nucleotides  Enzyme :- Ribonucleotide reductase  Co enzyme:- Thioredoxin (reduced form is converted to oxidized form) which requires NADP as a result elimination of water occurs in this reaction.

22. Purine catabolism  End product of purine catabolism is uric acid.  Normal blood levels: females:2-5mg/dl  Males: 3-7mg/dl  Daily excretion: 500- 700mg/dl.

23. Disorders of Purine Metabolism: DisorderDefect Comments Gout PRPP synthase/ Hyperuricemia HGPRT Lesch Nyhanlack of HGPRT Hyperuricemia syndrome SCID ADA High levels of dAMP von Gierke’s diseaseglucose -6-PTPase Hyperuricemia

24. GOUT (Gouty Arthritis): A defect of purine metabolism Serum Uric Acid Levels (mg/dl) Incidence of Gout (% of cases) Incidence of Gout (% of cases) >9.0 ~10% 7-9 0.5-3.5% <7.0 0.1% Guanine Xanthine Hypoxanthine Urate xanthine oxidase Allopurinol: a. decrease urate b. increase xanthine & hypoxanthine c. decrease PRPP Allopurinol: a. decrease urate b. increase xanthine & hypoxanthine c. decrease PRPP

25. Gout (hyperuricemia)  Primary: familial 1. Salvage pathway enzymes deficiency 2. Glucose -6 phosphatase deficiency (Von gierkes disease)  Secondary: 1. Rapidly growing malignant cells (leukemias) 2. Renal failure due to decreased excretion

26. Learning check  Why should Glucose -6 phosphatase deficiency (Von Gierke's disease) results in hyperuricemia and gout?

27. Clinical findings 1. Due to increased accumulation of uric acid as crystals in synovial fluid results in inflammation and arthritis. 2. At 30degrees the solubility of uric acid is lowered so it gets deposited around joints called tophi typical at metatarsophalangeal joint (big toe). 3. They can also be deposited in the kidneys causing renal calculi. 4. Treatment: Allopurinol compt.Inhibi of Xanthine oxidase decreases uric acid levels.

28. Gout

29. Adenosine deaminase (ADA) deficiency causes SCID (severe combined immunodeficiency),  Disorder that affects the immune system leading to malfunction resulting in open to infection from bacteria and viruses.  Caused by a mutation in a gene on chromosome 20.

30. Adenosine deaminase (ADA) deficiency  Without this enzyme, the body is unable to break down adenosine.  Lymphocytes contain high quantity of ADA.  Deficiency of ADA is manifested as reduced lymphocytes which leads to impaired cellular and Humoral immunity leading to infections.

31. How do people get ADA deficiency?  ADA deficiency is an autosomal recessive disorder.  Both parents need to pass the defective gene to their child in order for that child to inherit the deficiency.  Because ADA deficiency affects the immune system, people who have the disorder are more susceptible to all kinds of infections, particularly those of the skin, respiratory system, and gastrointestinal tract.  Sadly, most babies who are born with the disorder die within a few months. How do people get ADA deficiency? What are the symptoms of ADA deficiency?

32. How to diagnose ADA deficiency? Doctors can identify ADA deficiency during the mother's pregnancy: by taking a tiny sample of tissue from the amniotic sac where the baby develops (called chorionic villus sampling), by looking at enzyme levels in a fetal blood sample taken from the umbilical cord. After the child is born, doctors can test a sample of his or her blood to see if it contains ADA.

33. How to diagnose ADA deficiency?  PID :Test is now available to reduce the risk of passing on this genetic disorder, to children.  This test is called Preimplantation Genetic Diagnosis (PGD).  PGD tests an embryo for genetic defects and determines its sex prior to implantation.  Through this mechanism, the chance of having an affected child can be significantly reduced.

34. How is ADA deficiency treated? Gene therapy  The first person to receive gene therapy for ADA deficiency was four-year-old Ashanthi DeSilva.  The treatment was developed by three physicians—W. French Anderson, Michael Blaese, and Kenneth Culver.  DeSilva received her first treatment, an infusion of her own T cells implanted with normal ADA genes, on September 14, 1990 at the National Institutes of Health in Bethesda, Maryland.

35. How did DeSilva's T cells acquire the normal ADA genes?  retrovirus, (vectors) were made for carrying the normal ADA genes into the T cells.  The retrovirus vectors— carrying normal ADA genes— were mixed with T cells that had been extracted from DeSilva's blood and grown in culture dishes. The T cells were then infused back into DeSilva's blood where the normal ADA genes in them produced ADA. However, since T cells have a limited life span, DeSilva needed to receive periodic infusions of their genetically-corrected T cells.

36. Stem cell therapy  Subsequent research is focusing on developing a permanent cure for ADA deficiency using gene therapy.  Their own stem cells that had been implanted with normal ADA genes.  Stem cells live throughout the patient's life, and thus the patient should have a lifetime supply of ADA without requiring further treatment.

37. Purine salvage pathway Salvage pathways for the re-utilization of purines Nucleotides (purine and pyrimidine) are synthesized from intermediates in the degradative pathway for nucleotides. There are 2 salvage enzymes with different specificity; 1.Adenine phosphoribosyl transferase (APRT) 2. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) Salvage pathways for the re-utilization of purines Nucleotides (purine and pyrimidine) are synthesized from intermediates in the degradative pathway for nucleotides. There are 2 salvage enzymes with different specificity; 1.Adenine phosphoribosyl transferase (APRT) 2. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) Salvage pathway enzymes recycle 90% of purines. And also that are obtained from the diet.

38. Importance of salvage pathway  In tissues like RBC and brain where the de novo pathway is not operating.  This pathway economizes intracellular energy expenditure.

39. Substrates NucleosideEnzymeNucleotide Hypoxanthinehypoxanthine/gu anine phosphoribosyl transferase (HGPRT) IMP Guanine Adenine guanine phosphoribosyl t ransferase (HGPRT) phosphoribosyltr ansferase (APRT) GMP AMP

40. Food sources  Purines are found in high concentration in meat and meat products, especially internal organs such as liver and kidney. Plant based diet is generally low in purines.  Examples of high purine sources include: sweetbreads, anchovies, sardines, liver, beef kidneys, brains, meat extracts, herring, mackerel scallops.

41. Disorders of salvage pathway enzymes Lesch-Nyhan syndrome Is inherited as an X-linked trait. It mostly occurs in boys. Deficiency of HGPRT (hypoxanthine guanine phosphoribosyl transferase) The body needs this enzyme to recycle purines without it, abnormally high levels of uric acid build up in the body. The condition affects about 1 in 10,000 males.

42. Symptoms  Involuntary movements  Self mutilation: self-destructive behavior characterized by chewing off fingertips and lips  Mental retardation.  Hyperuricemia: gout-like swelling in some of their joints.  Renal UA crystals  Orange crystals in diapers  Often death Neurological manifestations suggest that the brain is dependent on the salvage pathway for the requirements of IMP and GMP.

43. Excess uric acid levels cause children to develop gout-like swelling in some of their joints.

44. Treatment  Investigations: Blood and urine tests may reveal high uric acid levels.  A skin biopsy may show decreased levels of the HGPPT enzyme.  No specific treatment exists for Lesch-Nyhan syndrome.  The gout medication allopurinol successfully decreases uric acid levels, but does not improve the neurological outcome.  Prognosis: The outcome is likely to be poor.

45. Prevention  Individuals with Lesch-Nyhan Syndrome and their families are recommended to undergo genetic counseling and testing.  Lesch-Nyhan Syndrome rarely occurs among females and female carriers typically do not show any symptoms but have a 50% chance of passing on the defective gene to her children.

46. Purine Metabolism Overview