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BLOOD Pyrimidine Nucleotides Cytidin E Uridine PRPP “De Novo”

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BLOOD Pyrimidine Nucleotides Cytidin E Uridine PRPP “De Novo” Dr. Mária Sasvári NUCLEOTIDE METABOLISM Metabolism of pyrimidine nucleotides 22 1 The structure of nucleobases N-containing, heterocyclic aromatic compounds; substituted purine or pyrimidine rings 22 RNA DNA 2 Pyrimidine nucleotides “de novo” BLOOD synthesis Catabolism CTP CMP cytidin e UTP UMP uridine salvage reactions PRPP CO2, NH3, b-alanine CoA 22 3 PYRIMIDINE nucleotide synthesis “de novo” UTP CTP synthesis salvage reactions UDP nucleosides UMP UMP-S bases Py 5,6 OA Py 4 H OA CAD 2 mito Py 1-3 Asp Gln Oroticaciduria 22 4 “de novo” PYRIMIDINE synthesis CO 2 Gln Carbamoyl-P synthetase ATP C 1. (cytoplasmic) ADP Py1 Glu NH2 C O O - P Carbamoyl phosphate Asp A Asp transcarbamoylase 2. Py2 22 5 “de novo” PYRIMIDINE synthesis COO - NH2 C O N COO- H carbamoyl-Asp D Dihydro-orotase 3. H2O Py3 O HN O N COO- H 22 6 Dihydro-orotate (H2OA) “de novo” PYRIMIDINE synthesis OA Py 4 NAD+ H2OA mito H2OA DEH 4. Py4 NADH O HN O N COO- H Orotate (OA) H2OA DEH = Dihydro-orotate dehydrogenase 22 7 “de novo” PYRIMIDINE synthesis PRPP OA-PRT 5. Py5 PPi O HN UMP-S (synthase) O N COO- R- P Orotidylate (OMP) Py6 OMP 6. CO2 decarboxylase OA-PRT = orotic acid (orotate) phosphoribosyl transferase OMP=orotidylate 22 UMP 8 UMP=uridylate UTP CTP UMP UDP ATP Pyrimidine monophosphate kinase 7. UMP ADP UDP ATP Pyrimidine diphosphate kinase 8. ADP UTP (2,4-dioxo) Gln ATP CTP synthetase 9. ADP + Pi Glu 22 CTP (2-oxo-4 amino9) Regulation of PYRIMIDINE “de novo” synthesis CAD CAD complex: Py 1-3 a single polypeptide three active centers + allosteric sites UTP + PRPP UMP 22 10 Orotic aciduria Main reasons of orotic aciduria 1. UMP-S deficiency Treatment: Oral uridine 2. Inhibition of UMP-S Allopurinol treatment (gout) 6-azauridine treatment (tumor) 3. Ornitine transcarbamoylase deficiency(urea cycle): NH3 CP Hyperammonemia, carbamoyl P(CP) accumulation CAD will use the leaking CP-t OA overproduction orotic aciduria 22 11 CATABOLISM CMP cytidine Nucleotide Nucleoside UMP uridine UMP (TMP) B CMP r -p 5’nucleotidase P i Pi B uridine (tymidine) r cytidine cytidine deaminase NH3 H2O 22 12 CO2, NH3, b-Alanine SALVAGE CMP citidin Nucleoside nucleotide UMP uridin uridine cytidine B ATP r ATP Uridine/cytidine kinase ADP ADP B r -p UMP CMP Cytidylate deaminase NH3 H2O 22 13 Deoxynucleotides P-P-O-H C 2 O B 3’ 2’ 22 14 The ribonucleotide reductase reaction (RR) NDP dNDP NDP: UDP/ADP/GDP/CDP H2O SH S RR RR SH S SH S TR: TR TR Tiredoxin Reductase SH S or: Glutaredoxin Reductase FADH2 FAD (glutathion) NADPH+ H+ NADP+ 22 15 REGULATION of RR: enzyme induction S-phase enzyme – active only in dividing cells! The RR is a dimer R1 – catalytic subunit constitutive expression R2 – regulatory subunit induced in the S-phase (E2F transcription factor) 22 16 Regulation of RR: Allosteric regulation 1. dATP: complete inhibition 2. Regulation of the ratio Substrates: Products: CDP dCDP dCTP dUDP dUMP UDP dTTP GDP + dGTP ADP + dATP 22 17 The thymidylate synthase reaction (TMP S) 5F-dUMP TMP S (colon cancer) dUMP dTMP 5 10 N Nmetylene H4F H2F 5N-metyl-H F 4 Ser,Gly Dihidrofolate reductase B 12 H4F NADPH+ H+ NADP+ Folate antagonists e.g. methotrexate tumor therapy 22 18 Inhibition of the folate synthesis: antibacterial effect Human: folate is a vitamin Bacteria: synthesis of folate could be selectively inhibited O H2N S N R O H sulphonamides 22 19 “Salvage” reaction of deoxynucleosides Deoxycytidine kinase (dCK) dCit dCMP limphoid specific ATP ADP Broad substrate specificity Substrates: dAde, dGua, dCit Analogues of deoxynucleotides: Antileucemic agents arabinosyl-cytozine 2-chloro-deoxyadenosine 22 20 “Salvage” reaction of deoxynucleosides Thymidine kinase (dTK) tymidine dTMP S-phase dependent enzyme ATP ADP Human dTK: narrow substrate specificity Herpes Simplex Virus : HSV- dTK: wide substrate specificity Selective inhibition of HSV – dTK with nucleoside analogues e.g. Antiviral medication (Aciclovir) e.g. HSV-dTK transfection into T lymphocytes: they might be selectively killed by Ganciclovir (AIDS – „Suicide gene therapy”) 22 21 22 22 SUMMARY „de novo” synthesis of purine- pyrimidine nucleotides „free” (no sugar) purine/pyrimidine ring NO YES (orotic acid) containing intermedier PRPP is an allosteric YES YES activator C1-TH4 donor YES NO (except thymidine!) role of Asp N donor incorporates origine of amino groups Asp, Gln Gln origine of the atoms in the ring Gly, Asp, Gln, CO2, Gln C1-TH4, CO2 Asp 22 23 SUMMARY „salvage” pathway of purine- pyrimidine nucleotides mainly from bases through PRPP reaction YES NO PRPP is necessary YES NO Mainly from nucleosides NO YES 22 24 SUMMARY Catabolic pathway of purine- pyrimidine nucleotides N of rings will be excreted YES NO Amino groups saved YES YES diseases hyperuricaemia orotic aciduria 22 25 SUMMARY: enzymes and diseases mentioned on the lecture Abbreviations: PRPP=phosphorybosyl pyrophosphate AMP= adenylate=adenosine monophosphate DE NOVO PYRIMIDINE SYNTHESIS GMP=guanylate=guanosine monophosphate CAD complex (REG): IMP=inosine monophosphate - Cytoplasmic carbamoyl phosphate synthetase XMP=xanthosine monophophate Aspartate transcarbamoylase OMP=orotidylate Dihydroorotase UMP=uridylate or uridine monophosphate Dihydroorotate dehydrogenase NDP = nucleoside diphosphate = ADP/GDP/CDP/UDP dNDP = deoxynucleoside diphosphate = dADP/dGDP/dCDP/dUDP UMP synthase complex (REG) H4F = tetrahydrofolate orotic acid (orotate) phosphoribosyl transferase H2F = dihydrofolate UMP-synthase REG: enzyme with allosteric regulation Pyrimidine monophosphate kinase Pyrimidine diphosphate kinase PURINE DE NOVO SYNTHESIS CTP synthetase PRPP synthetase (REG) Glutamine PRPP amidotransferase (REG) SYNTHESIS OF DEOXYRIBONUCLEOTIDES IMP dehydrogenase (REG) Ribonucleotide reductase, REG GMP synthase Thymidylate synthase Adenylosuccinate synthase (REG) Adenylosuccinase PYRIMIDINE CATABOLISM PURINE NUCLEOTIDE CYCLE 5’nucleotidase Adenylosuccinate synthase Cytidine deaminase Adenylosuccinase AMP deaminase PYRIMIDINE SALVAGE PURINE SALVAGE Uridine/cytidine kinase Adenine phosphorybosyl transferase Citidylate deaminase Hypoxanthine guanine phosphorybosyl transferase Thymidine kinase PURINE CATABOLISM Deoxycytidine kinase 5’nucleotidase Adenosine deaminase Diseases Purine nucleoside phosphorylase Orotic aciduria Anticancer drugs: 5-F-UMP, methotrexates Diseases Antibacterial drugs: sulphonamides Lesch-Nyhan syndrome Muscle adenosine deaminase (myoadenylate deaminase) deficiency Severe combined immunodeficiency (SCID) 22 26 Gout.
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