NUCLEOTIDE METABOLISM Metabolism of pyrimidine nucleotides Prof. Mária Sasvári NUCLEOTIDE METABOLISM Metabolism of pyrimidine nucleotides Gergely Keszler 2009.

The structure of nucleobases N-containing, heterocyclic aromatic compounds; substituted purine or pyrimidine rings RNA DNA

Pyrimidine nucleotides “de novo” synthesis BLOOD Catabolism cytidine uridine CTP  UTP   CMP    UMP  PRPP salvage reactions dTTP CO2, NH3, b-alanine  CoA

PYRIMIDINE nucleotide synthesis “de novo” synthesis UTP  UDP UMP CTP salvage reactions nucleosides UMP-S Py 5,6 OA H2OA mito Py 4 Asp Gln CO2 CAD Py 1-3

“de novo” PYRIMIDINE synthesis CO2 1. Glu Gln 2 ATP 2 ADP + Pi Carbamoyl-P synthetase II (CPSII) C Py1 NH2 C O O - P carbamoyl-P 2. Asp Asp transcarbamoylase A Py2

“de novo” PYRIMIDINE synthesis NH2 C O N H carbamoyl-Asp COO- COO - 3. Dihydroorotase D Py3 H2O HN O N H Dihydroorotate (H2OA) COO-

“de novo” PYRIMIDINE synthesis OA “de novo” PYRIMIDINE synthesis NAD+ 4. Dihydroorotate dehydrogenase NADH + H+ Py4 HN O N H orotate (OA) COO-

“de novo” PYRIMIDINE synthesis phosphoribosyl transferase 5. PPi PRPP Orotate phosphoribosyl transferase UMP-S (synthase) Py5 Py6 HN O N COO- R- P Orotidylate (OMP) 6. OMP decarboxylase CO2 UMP

Pyrimidine monophosphate Pyrimidine diphosphate UTP  UDP UMP CTP UMP UDP ADP ATP 7. Pyrimidine monophosphate kinase ADP ATP UTP (2,4-dioxo) 8. Pyrimidine diphosphate kinase Glu Gln CTP (2-oxo-4 amino) 9. CTP synthetase ADP ATP

PYRIMIDINE “de novo” synthesis Regulation of PYRIMIDINE “de novo” synthesis CAD Py 1-3 CAD complex: a single polypeptide three active centers + allosteric sites UTP PRPP + UMP

Comparison of CPS I and II isoenzymes CPSI CPSII • role urea cycle de novo pyrimidine biosynthesis • localization mitochondrial cytosolic • N-donor free NH3 glutamine • regulation + N-acetyl-glutamate + PRPP, - UTP

Orotic aciduria 1. UMP-S deficiency Main reasons of orotic aciduria treatment: oral uridine 2. Inhibition of UMP-S Allopurinol treatment (gout) 6-azauridine treatment (tumor) 3. Ornitine transcarbamoylase deficiency (urea cycle): NH3 CP  Type II hyperammonemia, carbamoyl P (CP) accumulation CAD can use the leaking CP  orotic acid overproduction  orotic aciduria

Nucleoside  nucleotide Uridine/cytidine kinase SALVAGE Nucleoside  nucleotide citidin uridin ® CMP UMP B r uridine cytidine ATP ADP ATP ADP Uridine/cytidine kinase B r -p UMP CMP Cytidylate deaminase H2O NH3

Nucleotide  Nucleoside CATABOLISM Nucleotide  Nucleoside cytidine uridine ® CMP UMP B r -p UMP (TMP) CMP 5’nucleotidase B r Pi uridine (thymidine) cytidine cytidine deaminase H2O NH3

cytidine deaminase pyrimidine nucleoside phosphorylase dihydropyrimidine dehydrogenase dihydropyrimidinase ureidopropionase Coenzyme A

ribonucleotide reductase enzyme Formation of deoxynucleotides NADP+ NADPH + H+ H2O β-D-ribofuranose (in RNA) 2’-deoxy-β-D-ribofuranose (in DNA) In cells, deoxyribonucleotides are synthesised from ribonucleotides by the ribonucleotide reductase enzyme

The ribonucleotide reductase reaction (RR) NDP dNDP NDP: UDP/ADP/GDP/CDP H2O RR S SH TR S SH TR: thioredoxin reductase or: glutaredoxin reductase (glutathion) FADH2 FAD NADPH+ H+ NADP+

Mechanism of the RR reaction ribonucleotide reductase

constitutive expression RR is active in dividing cells! The RR is a dimer R1 – catalytic subunit constitutive expression R2 – regulatory subunit induced in the S-phase RR is active in dividing cells!

Allosteric REGULATION 1. dATP: complete inhibition 2. Regulation of the ratio Substrates: CDP UDP GDP ADP Products: dCTP dTTP dGTP dATP + + dCDP  dUDP dUMP   

Synthesis of thymine nucleotides UMP UDP dUDP dUDP dTTP DNA Folate antagonists (chemotherapy!)

5-FU, a potent inhibitor of thymidylate synthase 5-fluoro-uracil (5-FU) inhibitor of thymine synthesis – WHY?? used to treat solid tumors (pancreas, lung, colon)

Inhibition of the folate synthesis: antibacterial effect Human: folic acid is a vitamin Bacteria: synthesis of folate can be selectively inhibited folic acid sulphonamides

The role of folate and vitamin B12 in DNA synthesis and cell division oxidative stress atherosclerosis  adenosine THF CH3-THF HomoCys Gly Ser SAM SAH -CH3 folate cycle B12 SAM/SAH cycle CH2-THF  dUMP  dTMP purine synthesis Cell division  Met    “methyl trap”  B12/folate deficiency: pernicious anemia

Folate deficiency – megaloblastic anemia

Vitamin B12 (cobalamin) deficiency Methylmalonic aciduria might be due to achlorhydria (chronic gastritis, no IF production) treatment: parenteral administration of B12 Methylmalonic aciduria Megaloblastic anemia (Pernicious anemia) nerve degeneration paralysis and death (B12 is needed for myelin synthesis) Anemia is due to impaired dTTP synthesis for DNA replication in hematopoetic precursor cells

“Salvage” reaction of deoxynucleosides Thymidine kinase (dTK) dThd  dTMP ATP ADP Deoxycytidine kinase (dCK) dCyd  dCMP lymphoid specific ATP ADP Broad substrate specificity Substrates: dAdo, dGuo, dCyd Analogues of deoxynucleotides: antileukemic agents purine and pyrimidine analogues

2-chloro-2’-deoxyadenosine Hairy cell leukemia (HCL) Purine analogues 6-mercaptopurine 8-azaguanine 2-chloro-2’-deoxyadenosine (CdA, Cladribine®) Hairy cell leukemia (HCL)

(araC; for treating AML) Pyrimidine analogues containing sugar derivatives 2’-deoxy-2’, 2’- -difluorocytidine (dFdC, Gemcitabine) 2’, 3’-dideoxy-cytidine arabinosyl-cytosine (araC; for treating AML)

“Salvage” reaction of deoxynucleosides Thymidine kinase (dTK) dThd  dTMP ATP ADP S-phase dependent enzyme Human dTK: narrow substrate specificity Herpes Simplex Virus : HSV- dTK: wide substrate specificity  Antiviral medication (Acyclovir) Suicide gene therapy (Gancyclovir, AIDS)

inhibition of viral DNA (RNA) replication Antiviral nucleoside analogues Acyclovir Stavudine Didanosine 9-hydroxyethoxy- methyl guanine, an acyclic nucleoside inhibition of viral DNA (RNA) replication herpes viruses, HIV inhibitors of retroviral reverse transcriptase

purine- pyrimidine nucleotides origin of the atoms in the ring „de novo” synthesis of purine- pyrimidine nucleotides „free” (no sugar) purine/pyrimidine ring containing intermedier NO YES (orotic acid) PRPP is an allosteric activator YES YES C1-TH4 donor YES NO (except thymidine!) role of Asp N donor incorporates origin of amino groups Asp, Gln Gln origin of the atoms in the ring Gly, Asp, Gln, C1-THF, CO2 Asp CO2, NH3 (Gln, Glu)

purine- pyrimidine nucleotides „salvage” pathway of purine- pyrimidine nucleotides mainly from bases through PRPP reaction YES NO PRPP is necessary YES NO mainly from nucleosides NO YES

purine- pyrimidine nucleotides Catabolic pathway of purine- pyrimidine nucleotides N of rings will be excreted YES NO amino groups saved YES YES diseases hyperuricemia orotic aciduria