Dr. Mária Sasvári

NUCLEOTIDE Metabolism of pyrimidine

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 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 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 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: 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:

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 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 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: and diseases mentioned on the lecture

Abbreviations: PRPP=phosphorybosyl AMP= adenylate= DE NOVO PYRIMIDINE SYNTHESIS GMP=guanylate= CAD complex (REG): IMP=inosine monophosphate - Cytoplasmic carbamoyl phosphate synthetase XMP=xanthosine monophophate Aspartate transcarbamoylase OMP=orotidylate 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 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 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