Nucleotides Synthesis and Degradation Javad Zavar Reza Ph
- Slides: 93
Nucleotides: Synthesis and Degradation Javad Zavar Reza Ph. D in Clinical Biochemistry Department of Biochemistry School of Medicine 1
Nitrogenous Bases Planar, aromatic, and heterocyclic Derived from purine or pyrimidine Numbering of bases is “unprimed” 2
Nucleic Acid Bases Purines Pyrimidines 3
Sugars Pentoses (5 -C sugars) Numbering of sugars is “primed” 4
Sugars D-Ribose and 2’-Deoxyribose *Lacks a 2’-OH group 5
Nucleosides 6
Phosphate Groups Mono-, di- or triphosphates Phosphates can be bonded to either C 3 or C 5 atoms of the sugar 7
Nucleotides Result from linking one or more phosphates with a nucleoside onto the 5’ end of the molecule through esterification 8
Nucleotides Monomers for nucleic acid polymers Nucleoside Triphosphates are important energy carriers (ATP, GTP) Important components of coenzymes – FAD, NAD+ and Coenzyme A 9
Naming Conventions Nucleosides: – Purine nucleosides end in “-sine” Adenosine, Guanosine – Pyrimidine nucleosides end in “-dine” Thymidine, Cytidine, Uridine Nucleotides: – Start with the nucleoside name from above and add “mono-”, “di-”, or “triphosphate” Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine Diphosphate 10
Nucleotide Biosynthesis De novo Biosynthesis Salvage Biosynthesis 11
Nucleotide Metabolism PURINE RIBONUCLEOTIDES: De novo – i. e. , purines are not initially synthesized as free bases – First purine derivative formed is Inosine Mono-phosphate (IMP) 12
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Steps 1 Step 1: Activation of ribose-5 -phosphate – product: 5 -phosphoribosyl-a-pyrophosphate (PRPP) – PRPP is also a precursor in the biosynthesis of: pyrimidine nucleotides Histidine Tryptophan 14
Step 1: purine synthesis 15
Step 2: purine synthesis: commited step 16
Step 3 : purine synthesis 17
Acquisition of purine atom C 8 & purine atom N 3 18
Step 6: purine synthesis 19
Step 7: purine synthesis Acquisition of C 6 introduced as HCO 3 - 20
Steps 8 thru 11 Step 8: acquisition of N 1 – N 1 is acquired from aspartate in an amide condensation reaction – enzyme: SAICAR synthetase – product: 5 -aminoimidazole-4 -(Nsuccinylocarboxamide)ribotide (SAICAR) – reaction is driven by hydrolysis of ATP 21
Step 8: purine synthesis 22
Step 9: purine synthesis 23
Step 10: purine synthesis 24
Step 11 cyclization or ring closure to form IMP water is eliminated in contrast to step 6 (closure of the imidazole ring), this reaction does not require ATP hydrolysis once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells 25
Step 11: purine synthesis 26
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Synthesis of adenine and guanine nucleotides 28
Purine nucleoside diphosphates and triphosphates: - to be incorporated into DNA and RNA, nucleoside monophosphates (NMP’s) must be converted into nucleoside triphosphates (NTP’s) - nucleoside monophosphate kinases (adenylate & guanylate kinases) - nucleoside diphosphate kinase 29
REGULATION OF PURINE NUCLEOTIDE BIOSYNTHESIS 30
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Salvage of Purines Adenine phosphoribosyltransferase (APRT) 32
Salvage of Purines Salvage is needed to maintain the purine pool (biosynthesis is not completely adequate, especially in neural tissue) Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) Hypoxanthine + PRPP IMP + Ppi Guanine + PRPP GMP + Ppi Lack of HGPRT leads to Lesch-Nyhan syndrome. Lack of enzyme leads to overproduction of purines which are metabolized to uric acid, which damages cells 33
Salvage of purines Salvage of purine bases 34
Lesch-Nyhan syndrome there is a defect or lack in the HGPRT enzyme the rate of purine synthesis is increased about 200 X uric acid level rises and there is gout in addition there are mental aberrations patients will self-mutilate by biting lips and fingers off 35
Lesch-Nyhan syndrome 36
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Purine Autism 25% of autistic patients may overproduce purines To diagnose, must test urine over 24 hours – Biochemical findings from this test disappear in adolescence – Must obtain urine specimen in infancy, but it’s difficult to do! 38
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Origin of atoms in pyrimidine ring 40
Step 1: synthesis of carbamoyl phosphate Condensation of glutamine, bicarbonate in the presence of ATP Carbamoyl phosphate synthetase exists in 2 types: CPS-I which is a mitochondrial enzyme and is dedicated to the urea cycle and arginine biosynthesis) and CPS-II, a cytosolic enzyme used here 42
Step 1: pyrimidine synthesis CPS-II is the major site of regulation in animals: UDP and UTP inhibit the enzyme and ATP and PRPP activate it It is the committed step in animals 43
Step 2: synthesis of carbamoyl aspartate enzyme is aspartate transcarbamoylase (ATCase) catalyzes the condensation of carbamoyl phosphate with aspartate with the release of Pi ATCase is the major site of regulation in bacteria; it is activated by ATP and inhibited by CTP carbamoyl phosphate is an “activated” compound, so no energy input is needed at this step 44
Step 2: pyrimidine synthesis 45
Step 3: ring closure to form dihydroorotate enzyme: dihydroorotase forms a pyrimidine from carbamoyl aspartate water is released in this process 46
Step 3: pyrimidine synthesis 47
Step 4: oxidation of dihydroorotate to orotate an irreversible reaction enzyme: dihydroorotate dehydrogenase oxidizing power is derived from quinones (thru coenzyme Q) 48
Step 4: pyrimidine synthesis 49
Step 5: pyrimidine synthesis 50
Step 6: pyrimidine synthesis 51
The big picture again 52
Orotic aciduria an inherited human disease caused by a deficiency in the multifunctional enzyme that catalyzes the last 2 steps in the pyrimidine synthesis large amounts of orotic acid in urine retarded growth and severe anemia treat by administration (injection) of uridine and/or cytidine 53
Leflunomide (Arava) Leflunomide is an isoxazole immunomodulatory agent which inhibits dihydroorotate dehydrogenase) and has antiproliferative activity. Several in vivo and in vitro experimental models have demonstrated an anti-inflammatory effect. 54
Leflunomide (Arava) 55
Synthesis of uridine and cytidine triphosphate (in bacteria, ammonia donates the amino group) 56
Regulation of pyrimidine nucleotide biosynthesis UTP and CTP are feedback inhibitors of CPS II 57
Formation of deoxyribonucleotides d. ADP, d. GDP, d. UDP and d. CDP are all synthesized by the same enzyme Synthesized from nucleoside diphosphate (not mono or triphosphate) by ribonucleotide reductase 58
Synthesis of d. TMP Methylation of d-UMP via N 5, N 10 -methylene THF Reaction inhibited by 5 -fluorouracil (Efudex) 59
Activation of 5 -fluorouracil 60
Regeneration of N 5, N 10 Methylen THF d. UMP d. TMP thymidylate synthase DHF N 5, N 10 – METHYLENE-THF NADPH + H+ GLYCINE dihydrofolate reductase serine hydroxymethyl transferase NADP+ SERINE THF
Inhibitors of N 5, N 10 Methylene THF Regeneration METHOTREXATE AMINOPTERIN TRIMETHOPRIM d. UMP d. TMP DHF thymidylate synthase N 5, N 10 – METHYLENE-THF Fd. UMP NADPH + H+ GLYCINE serine hydroxymethyl transferase SERINE THF dihydrofolate reductase NADP+
Hydroxyurea (Hydrea) inhibits the enzyme ribonucleotide reductase – – – DNA synthesis cannot occur Cell are killed in the S phase Drug holds other cells in the G 1 phase Primarily used to treat chronic myelogenous leukemia Cancer cell develop resistance by: – – increasing quantity of inhibited enzyme decreasing sensitivity of enzyme for inhibitor used orally major side effect is leukopenia 63
Another inhibitor of ribonucleotide reductase: indicated for non-small cell lung cancer (usually with cisplatin) also first line treatment for non-resectable pancreatic cancer 64
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Degradation of AMP 68
PENTOSTATIN previously called deoxycoformycin (DCF) a purine analog with a 7 -membered-ring potent inhibitor of adenosine deaminase ADA is a key enzyme which regulates adenosine levels in cells indicated for refractory hairy cell leukemia other uses: chronic lymphocytic leukemia and lymphomas 69
ADA deficiency In the absence of ADA lymphocytes are destroyed deoxyadenosine is not destroyed, is converted to d. AMP and then into d. ATP is a potent feedback inhibitor of deoxynucleotide biosynthesis this leads to SCID (severe combined immunodeficiency disease) Infants with this deficiency have a high fatality rate due to infections 70
ADA deficiency treatment consists of administering pegylated ADA which can remain in the blood for 1 – 2 weeks more efficient is gene therapy: replacing the gene that is missing or defective gene therapy has been performed on selected patients 71
Degradation of GMP and XMP 72
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CATABOLISM OF PURINES 74
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GOUT Associated with abnormal amounts of urates in the body Early stage: recurring acute non- articular arthritis Late stage: chronic deforming polyarthritis and eventual renal complication Disease with rich history dating back to ancient Greece 76
GOUT Once fashionable to associate gout with intelligence People with gout: – Isaac Newton – Benjamin Frankin – Martin Luther – Charles Darwin 77
Gout prevail mainly in adult males rarely encountered in premenopausal women symptoms are cause by deposition of crystals of monosodium urate monohydrate (can be seen under polarized light) usually affect joints in the lower extremities (the big toe is the classic site) 78
Gout 79
Diagnostic features usually affect joints in the lower extremities ( 95%) onset is fast and sudden pain is usually severe; joint may be swollen, red and hot attack may be accompanied by fever, leukocytosis and an elevated ESR 80
Drugs which may induce hyperuricemia niacin thiazides and other diuretics low dose aspirin pyrazinamide ethambutol cyclosporine cytotoxic drugs 81
Non-pharmacological approaches Avoid purine rich foods: – red meat and organ meat (liver, kidneys) – shellfish, anchovies, mackerel, herring – meat extracts and gravies – peas and beans, aspargus, lentils – beer, lager, other alcoholic beverages Weight loss Control alcohol (binge drinking) 82
Pharmacological management of gout based on the premise that the hyperuricemia is due to both overproduction and underexcretion of uric acid symptomatic relief of pain is also achieved with analgesics (i. e. indomethacin) drugs used: – analgesics (NSAIDs) – uricosuric agents – xanthine oxidase inhibitors 83
Colchicine a non-basic alkaloid from the seeds and corms of Colchicum autumnale (Meadow Safron) 84
COLCHICINE used in the symptomatic treatment of acute attacks of gout decreases leukocyte motility, decreases phagocytosis and lactic acid production not used in other forms of arthritis a very potent drug can cause severe GI distress and abdominal pain 85
Inhibits the tubular reabsorption of uric acid Inhibit the tubular excretion of certain organic acid via the transporter Also used to enhance plasma concentration of certain anti-infective (beta lactams) 86
ALLOPURINOL (Zyloprim) prevention of attacks of gouty arthitis and nephropathy also used during chemotherapy of cancer and to prevent recurrent calcium oxalate calculi metabolized to oxypurinol (also an inhibitor of xanthine oxidase) inhibits the metabolism of certain anticancer drugs (6 -MP, azathioprine) 87
An inhibitor of xanthine oxidase; prevents the formation of uric acid from precursorial purines 88
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Catabolism of a pyrimidine 90
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