Questions on Purine and Pyrimidine Metabolism:

Transcription

1 Questions on Purine and Pyrimidine Metabolism: 1. Mention the Origin of Carbon and itrogen Atom in Purine Ring. (2) 2. Sources of various atoms of purine ring. (4) 3. Give an account on salvage pathway. (5) 4. Describe two reactions of salvage pathway of purine nucleotide synthesis (2) 5. Purine salvage pathways (5) 6. Catabolism of purines (5) 7. Synthesis of Uric acid. (5) 8. Gout. (5) 9. Mention any two causes of hyperuricemia (2) 10. What is gout? Mention two causes of gout. (2) 11. ame the enzyme defect in: Primary Gout. (3 or 4) 12. ame the biochemical defect in Gout. (2) 13. Allopurinol. (2) 14. Describe the causes and features of hyperuricemia. Mention the drugs to lower plasma uric acid level. (2+2+1 =5) 15. What is normal serum uric acid level? ame two pathological conditions associated with hyperuricemia. (2) 16. Mention the causes and features of Lesch -yhan syndrome. (2) 17. Lesch-yhan syndrome & Orotic aciduria. (5 ) 1

2 Purine and Pyrimidine ucleotide Metabolism Purine Bases H 2 O H H 2 H Adenine H Guanine IntermediateEnd Product of in Purine Catabolism Purine Catabolism O O H Hypoxanthine Pyrimidine Bases O H H O H Uric Acid H 2 O H O O H H Cytosine Uracil CH 3 O H O H Thymine 2

3 ucleoside (Base + Sugar) H 2 HOH 2 C OH OH OH ucleotide (Base + Sugar + Phosphate) Adenosine (Adenine + Ribose) H 2 P PP OH 2 C OH OH OH Adenosine triphosphate (ATP) (Adenine + Ribose + 3 Phosphate groups) Functions of ucleotides Ribonucleoside and deoxyribonucleoside phosphates (nucleotides) are essential for all cells for synthesis of DA and RA and therefore, for cell proliferation and for synthesis of proteins. In addition, nucleotides, particularly ATP, play an important role as energy currency in the cell. ucleotides also serve as carriers of activated intermediates (e.g. UDP-glucose, S- adenosyl methionine, etc) in the synthesis of some carbohydrate, lipid and proteins. ucleotides are structural components of a number of coenzymes such as AD +, ADP +, FAD and coenzyme A. Finally nucleotides are important regulatory compounds (camp, cgmp) for many of the pathways of intermediary metabolism, inhibiting or activating key enzymes. Overview of Purine and Pyrimidine ucleotide Metabolism Metabolism of purine and pyrimidine nucleotides includes their biosynthesis and catabolism. 3

4 The purine and pyrimidine nucleotides can be synthesized 1) de novo (de novo = from scratch), or can be obtained through 2) salvage pathways, by reutilization of free bases and nucleosides resulting from normal cell turnover or from the diet Amino acids, CO 2, and ribose-5-phosphate (from the HMP shunt pathway) serve as sources for carbon, nitrogen and oxygen atoms of purine and pyrimidines nucleotides. Overview of Purine and Pyrimidine ucleotide Biosynthesis Ribose 5-phosphate (from HMP shunt pathway), Amino acids, CO 2, FH 4 De novo Synthesis (Synthesis from Scratch) Salvage Pathways Mainly in the Liver PiPi P-Ribose-PiPi (PRPP) Base (Purine & Pyrimidine Bases) MPBase-ribose (Ribonucleoside monophosphate) ADP ATP (ucleosides) ATP ADP Reduction O ATP ADP DPdDPdTP ATP (For DA Synthesis) TP ADP 4

5 In case of de novo biosynthesis of purine nucleotides, the purine ring is built upon a preexisting ribose 5-phosphate, whereas pyrimidine ring is synthesized before being attached to ribose 5-phosphate. De novo Synthesis of Purine ucleotide Ribose 5-phosphate Amino acids De novo Synthesis of Pyrimidine ucleotide CO 2, Amino acids FH 4 CO 2 Pyrimidine Base Ribose 5-phosphate Purine ucleotide Pyrimidine ucleotide Dietary uptake of purine and pyrimidine bases is low, because most of the ingested nucleic acids are metabolized by the intestinal epithelial cells. The end product of purine catabolism in humans is uric acid. Unlike the purine rings, which are not cleaved in human cells, the pyrimidine ring can be opened and degraded to highly water-soluble compounds. Medical Importance of ucleotide Metabolism Defects in the metabolic pathways for de novo synthesis or salvage of nucleotides result in clinical diseases or syndromes. Defects in degradation of nucleotides also lead to clinical problems. These include gout (defect in de novo purine nucleotide synthesis), Lesch-yhan syndrome (defect in purine salvage pathway), orotic aciduria (defect in de novo pyrimidine nucleotide synthesis) and immunodeficiency diseases (defects in purine nucleoside degradation). Because nucleotide synthesis is required for DA replication and RA synthesis in dividing cells, drugs that block de novo pathways of nucleotide synthesis have been used successfully as antitumor and antiviralagents. Purine ucleotide Metabolism Contents: Biosynthesis. De novo synthesis Salvage pathways Catabolism 5

6 De novo Synthesis of Purine ucleotides The de novo pathway of purine synthesis is complex, consisting of eleven steps and requiring six molecules of ATP for every purine synthesized. The purine ring is built upon a pre-existingribose 5-phosphate (synthesized in the HMP shunt pathway) by a series of reactions adding carbon and nitrogen atoms. The precursors that donate the component to produce purine nucleotides include Ribose 5-phosphate, glutamine, glycine,aspartate, carbon dioxide and 10 -formyl tetrahydro folate (FH 4 ) Sources of Individual Atoms in Purine Ring 6 Aspartate 15C7 CO 2 C Glycine 8C 5, 10 Methenyl C24C 9 tetrahydrofolate 3 10 formyl H Tetrahydrofolate Amide nitrogen of glutamine Many compounds contribute to the atoms of purine ring of the nucleotides. 1 of purine is derived from amino group of Aspartate. C 2 arisefrom 10 formyl THF. 3 and 9 are obtained from amide group of Glutamine (Gln). C 4, C 5 & 7 are contributed by Glycine. C 6 directly comes from CO 2. - C 8 arise from 5 10 Methenyl THF Tissues: The purine nucleotides are synthesized by most of the tissues. However the major site is Liver. Activity of the pathway is low in brain and absent in RBCs. Subcellular Site: Cytoplasm 6

7 Reactions of the Pathway HMP Shunt Pathway PRPP Synthetase Ribose-5-phosphate ATP Mg 2+ AMP Glutamine PRPP Amidotransferase De novo Synthesis of Pyrimidine ucleotides and Purine & Pyrimidine Salvage Pathways Phosphoribosyl pyrophosphate (PRPP) Glutamine Glutamate Amino acids 5'-Phosphoribosylamine FH 4 CO 2 IMP Kinase Kinase AMP GMP Oxidative Phosphorylation & Substrate Level Phosphorylation ATP ADP GTP GDP Kinase Ribose-5-phosphate, produced in the HMP shunt pathway, is the starting material for purine nucleotide synthesis. Phosphoribosyl pyrophosphate (PRPP) an intermediate (synthesized in the first step) is of major significance in nucleotide metabolism. P OH 2 CH O P P OH OH Phosphoribosyl pyrophosphate (PRPP) 7

8 PRPP is required in -- De novo synthesis of Pyrimidine and Purine ucleotides -- The Salvage Pathways for Purine and Pyrimidine ucleotides -- Biosynthesis of ucleotide Coenzymes The purine ring is assembled on the ribose-5-phosphate moiety of PRPP. Glutamine PRPP amidotransferase transfers the amide group of glutamine (Gln)to C 1 of PRPP forming 5-phosphoribosyl-1-amine. This reaction is the committed step in purine nucleotide biosynthesis. This is then followed by a series of reactions leading to the synthesis of IMP. Inosine monophosphate (IMP), containing hypoxanthine as the base, is the parent nucleotide synthesized in the de novo pathway.amp and GMP are in turn formed from IMP. PRPP synthetaseand glutamine PRPP amidotransferase catalyzing the first two steps are the regulatory enzymes for the pathway. Regulation 1. The intracellular concentration of PRPP (substrate availability) regulates purine synthesis to a large extent. That is, the rate of nucleotide synthesis is directly proportional to concentration of PRPP. 2. The activities of PRPP synthetase,glutamine PRPP amidotransferase and enzymes catalyzing synthesis of AMP and GMP from IMP are regulated by the end products, namely, purine ribonucleotides AMP, ADP, ATP, GMP, GDP and GTP, by allosteric mechanism. Purine Salvage Pathways Meaning/Definition Free purine basesand nucleosidesthat result from the normal turnover of cellular nucleic acids and nucleotides or that are obtained from the diet can be reutilized/recycled/salvagedto nucleotidesby the so-called salvage pathways. Importance Salvage pathway requires far less energy than de novo synthesis The salvage pathway is particularly important in certain tissues such as brain and RBCs where de novo synthesis of purine nucleotides is not active. 1. Salvage of Purine Bases Purine bases that are salvaged are adenine, guanine and hypoxanthine to their respective nucleotides. 8

9 Phosphoribosyl pyrophosphate (PRPP) provides the ribose -5 phosphate group. There are two specific enzymes that catalyze the transfer of the ribose phosphate from PRPP to free purine bases 1) hypoxanthineguaninephosphoribosyl transferase (HGPRTase)and 2) adenine phosphoribosyl transferase (APRTase). 1. Hypoxanthine-guaninephosphoribosyl transferase (HGPRTase)catalyzes the formation of nucleotides from hypoxanthine or guanine Hypoxanthine IMP PRPP PPi Guanine GMP PRPP PPi 2. Adenine phosphoribosyl transferase (APRTase)catalyzes the formation of AMP from adenine. Adenine AMP PRPP PPi Salvage of Purine ucleosides Kinase Purine ribonucleosidepurine ribonucleoside monophosphate ATP E.g. Guanosine kinase GuanosineGMP ADP ATP ADP 9

10 Catabolism of Purine ucleotides Formation of Uric Acid Degradation of purine nucleotides mainly occurs in the liver. Uric acid is the end product of purine metabolism in humans. Purine rings are not cleaved in humans. AMP deaminase AMP IMP GMP H 2 O H 3 H 2 O H 2 O H 2 O ucleotidase ucleotidaseucleotidase Pi Pi Pi Adenosine deaminase Adenosine Inosine Guanosine H 2 O H 3 Pi Pi Purine nucleoside phosphorylase Ribose-1-phosphate Hypoxanthine Purine phosphorylase nucleoside H 2 O + O 2 Xanthine oxidase Ribose-1-phosphate H 2 O 2 Guanase Xanthine H 3 H 2 O Guanine H 2 O + O 2 H 2 O 2 Xanthine oxidase Mo, Fe URIC ACID 10

11 et excretion of total uric acid in normal humans is mg/day. ormal blood level of uric acid in males 3 9 mg/dl and in females mg/dl Disorders of Purine Metabolism These include gout,lesch-yhan syndrome and immunodeficiency diseases. Hyperuricemia and Gout The most common abnormality of purine metabolism is an elevation of uric acid level in blood, referred to as hyperuricemia. All the clinical manifestations of hyperuricemia are due to low water-solubility of uric acid. Hyperuricemia may clinically manifest as arthritis calledgout or renal stone formation. Causes of Hyperuricemia (Gout) The causes may be classified as primary and secondary. Primary causes due to genetic defect leading to overproduction of uric acid Secondary causes secondary to other diseases Primary Causes This is mostly related to increased synthesis of purine nucleotides, which are degraded leading to overproduction of uric acid. Following are the important metabolic defects associated with hyperuricemia and gout. (i) Over activity of enzyme PRPP synthetase (ii) Over activity of enzyme glutamine PRPP amidotransferase (iii) Partial deficiencyhgprtase HGPRTase deficiency causes increased synthesis of purine nucleotides by 2 ways 1) decreased utilization of purines by salvage pathway results in the accumulation and diversion of PRPP for purine nucleotide synthesis; 2) the defect in salvage pathway leads to decreased levels of IMP and GMP causing impairment in the feed back inhibition of their production. (iv) Glucose-6-phosphatase deficiency (Von-Gierke s disease): In this conversion of glucose-6-phosphate to glucose is blocked. Glucose-6-phosphate is thus channeled into HMP shunt pathway resulting in elevated levels of ribose-5-phosphate, PRPP, purine nucleotides and ultimately overproduction of uric acid. Secondary Causes This is due to various diseases causing eitherincreased synthesis ordecreased excretion of uric acid. (i) (ii) Increased production of uric acid due to enhanced turnover rate of nucleic acids is observed in various cancers (leukemia), polycythemia, psoriasis, etc and also in starvation (raised rate of catabolism) and trauma. Reduced excretion rate may occur inrenal failure. 11

12 Causes of Hyperuricemia (Gout) Glucose-6-phosphatase (Von Gierke s disease) Glucose-6-phosphate Glucose HMP Shunt Pathway Primary Causes PRPP Synthetase Ribose-5-phosphate PRPP Glutamine PRPP Amidotransferase 5'-Phosphoribosylamine IMPHypoxanthine HGPRTase AMPGMPGuanine Uric Acid (Hyperuricemia) Secondary Causes Increased Production of Uric AcidReduced Renal Excretion (Due to enhanced turnover rate of nucleic acids) Renal failure Cancers (leukemia), Polycythemia, Psoriasis, Starvation (raised rate of catabolism) andtrauma. Clinical Manifestations of Hyperuricemia/Gout 1. At physiological ph, uric acid is found in a more soluble form as sodium urate. 2. In hyperuricemia, serum urate levels exceed the solubility limit resulting in crystallization of sodium urate in soft tissues and joints in the form of deposits called tophi. 3. This causes inflammation in the joints resulting in painful gouty arthritis. 4. Uric acid may also precipitate in kidney tubules and ureters that result in stone formation (calculi) and renal damage. Treatment Allopurinolis the drug administered to treat gout. 12

13 It is a structural analog of hypoxanthine, therefore inhibitsxanthine oxidase and thereby decreases formation of uric acid. Probenecidmay be administered toiincrease renal excretion of urateby decreasing its reabsorption from renal tubules. Gouty attacks may be precipitated by high purine diet (animal foods) and increased intake of alcohol. Therefore these must be restricted in the diet. Lesch-yhan Syndrome Cause:CompleteDeficiency of HGPRTase enzyme. Inheritance pattern: X-linked, recessive Biochemical Finding: Hyperuricemia Clinical Manifestations: Uric acid renal stones, gout eurological abnormalities such as mentalretardation, self-mutilation. Biochemical Basis: HGPRTase deficiency results in decreased utilization of purines by salvage pathway, ultimately leading to increased synthesis and degradation of purines. eurological symptoms may be related to dependence of brain on the salvage pathway for biosynthesis of purine nucleotides. Adenosine Deaminase Deficiency(Severe combined immunodeficiency disease or SCID) Adenosine deaminase deficiency is associated with a severe combined immunodeficiency disease in which both thymus-derived lympocytes (T cells) and bone marrow-derived lympocytes (B cells) are sparse and dysfunctional causing severe immunological dysfunction. 13

14 Pyrimidine ucleotide Metabolism Pyrimidine nucleotide metabolism includes biosynthesis and catabolism. Biosynthesis includes de novo biosynthesis and salvage pathways. Overview of Pyrimidine ucleotide Metabolism De novo Synthesis CO 2, Amino acids Ribose 5-phosphate Pyrimidine Base Pyrimidine ucleotides Salvage Pathway Pyrimidine Bases Catabolism CO 2, H 3, -alanine and -aminoisobutyrate De novo Biosynthesis of Pyrimidine ucleotides Pyrimidine and purine nucleotide biosynthesis share several common precursors: PRPP, glutamine, CO 2, aspartate and, for thymine nucleotide, tetrahydrofolate In case of de novo biosynthesis of pyrimidine nucleotides, the pyrimidine ring is synthesized before being attached to ribose-5-phosphate (whereas purine ring is built upon a pre-existing ribose 5-phosphate). Sources of Individual Atoms in Pyrimidine Ring Amide nitrogen 4 of Glutamine C C 5, 10 -MethyleneTetrahydrofolate 3 C 5(for synthesis of TMP) CO 2 2 C C6Aspartic acid 1 14

15 C 2 of the pyrimidine ring is derived from CO 2. 3 is derived from amide nitrogen of glutamine. 1, C 4, C 5 and C 6 are derived from aspartate. The methyl group attached to C 5 in thymine is derived from 5, 10 -Methylene tetrahydrofolate Reactions of the Pathway Overview of the Pathway Glutamine + CO 2 Carbamoyl phosphate Orotic acid PRPP Uridine monophosphate (UMP) Orotate monophosphate (OMP) UDP dump dtmp UTP CTP UMP is the first pyrimidine ribonucleotide to be synthesized. All other pyrimidine nucleotides are synthesized from UMP. UMP is converted to UDP, which serves as precursor for the synthesis of dump, dtmp, UTP and CTP. 15

16 Glutamine Carbamoyl phosphate synthetase-ii Glutamate (CPS-II) CO 2 Carbamoyl phosphate Aspartate transcarbamoylase 2 ATP 2 ADP + Pi Aspartate DihydroorotatePi dehydrogenase Dihydroorotase Orotic DihydroCarbamoyl aspartate acid Orotate PRPP H 2 O Orotate Phosphoribosyl transferase PPi OMP (orotidine monophosphate) Decarboxylase UMP CO 2 ATP Kinase ADP Ribonucleotide reductase UDP dudp ATP Kinase H 2 O ADP Pi UTP dump ATP Glutamine 5, 10 -Methylene tetrahydrofolate ADP GlutamateDihydrofolate Thymidylate synthase CTP dtmp 16

17 Regulation CPS-II is the regulatory enzyme of pyrimidine synthesis. It is allostericallyactivated by PRPP and ATP and inhibited by UTP. Catabolism of Pyrimidine ucleotides Catabolism of pyrimidine produces water-soluble metabolites. Catabolism of Cytosine and Uracil yields -alanine, CO 2, and H 3 Thymine on catabolism produces -aminoisobutyrate,co 2 and H 3. Pseudouridine is excreted unchanged. Disorders of Pyrimidine ucleotide Metabolism Since the end products of pyrimidine catabolism are highly water soluble, clinically detectable abnormalities due to pyrimidine overproduction are few. Orotic Aciduria Orotic aciduriais caused due to genetic defect in the enzymes of de novo biosynthesis of pyrimidine nucleotides Genetic defects cause orotic aciduria Type I and Type II. In Type I there is deficiency of both orotatephosphoribosyl transferaseandorotidylatedecarboxylase. Orotic aciduria TypeII results from deficiency of only orotidylate decarboxylase. Both Type I and Type II are characterized by excretion of orotic acid in urine, severe anemia andretarded growth. Feeding diet rich in uridine or cytidine is an effective treatment for orotic aciduria. These compounds provide pyrimidine nucleotides required for DA and RA synthesis. Besides this, UTP inhibits CPS-II and blocks synthesis of orotic acid. Inhibitors of Purine and Pyrimidine ucleotide Synthesis Inhibitors of de novo biosynthesis of purine and pyrimidine nucleotides are structural analogs of substrates/ intermediates of these pathways and inhibit specific enzymes of the pathways by competitive inhibition. These inhibitors by inhibiting purine and pyrimidine synthesis can slow down DA replication in mammalian cells. These compounds can, therefore, arrest the growth of tissue where cell division is rapid. These inhibitors, thus, are useful as anticancer and antiviral drugs.these drugs, however, are toxic to all other rapidly dividing normal cells such as foetal tissues, bone marrow, skin, gastro intestine tract or hair follicles and can cause anemia, scaly skin, GI tract disturbance and baldness. 17

18 1. 6-Mercaptopurine andthioguanine are purine analogs that are used clinically in the treatment of some leukemias. 2. Structural analogs of folic acid (folate)competitively inhibit folate reductase,thus interfering with the synthesis of coenzyme tetrahydrofolate (THF). DihydrofolateDihydrofolate reductase Folate reductase DihydrofolateTetrahydrofolate Tetrahydro folate is required for both purine and pyrimidine nucleotide synthesis. Methotrexateis ananalog of folic acid and is used as anticancer drug. Sulfonamides are structural analogs of para amino benzoic acid (PABA) that is required for the synthesis of folic acid in bacteria, hence their use as antibacterial drugs. Humans cannot synthesis folic acid and must rely on external sources of this vitamin. Therefore, sulfa drugs do not interfere with human purine synthesis 3. Azaserine and diazonorleucineare glutamine analog and inhibit the enzymes that utilize glutamine as amino group donor in the biosynthesis of purine nucleotides Fluorouracil and 5-iodo-2-deoxyuridine are anti cancer drugs, which competitively inhibit thymidylate synthase (enzyme of TMP synthesis). 18