1

NUCLEOTIDE METABOLISMNUCLEOTIDE METABOLISMMetabolism of pyrimidine nucleotidesMetabolism of pyrimidine nucleotides

NUCLEOTIDE METABOLISMNUCLEOTIDE METABOLISMMetabolism of pyrimidine nucleotidesMetabolism of pyrimidine nucleotides

ProfProf. Mária Sasvári. Mária Sasvári

Gergely Keszler2009.

2

The structure of nucleobases

N-containing, heterocyclic aromatic compounds; substituted purine or pyrimidine rings

RNA DNA

3

BLOOD

Pyrimidine nucleotidesPyrimidine nucleotidesPyrimidine nucleotidesPyrimidine nucleotides

cytidine

uridine

PRPP

“de novo” synthesis

CTP

UTP

Catabolism

CO2, NH3, -alanine

CoA

salvagereactions

CMP

UMP

dTTP

4

UTP

UDP

UMP

CTP“de novo” synthesis

AspGlnCO2

CADPy 1-3

H2OAmito

Py 4

UMP-SPy 5,6 OA

PYRIMIDINE nucleotide synthesisPYRIMIDINE nucleotide synthesisPYRIMIDINE nucleotide synthesisPYRIMIDINE nucleotide synthesis

nucleosides

salvagereactions

5

CO2

2.

Asp

““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis

1.

Glu

Gln2 ATP

2 ADP + Pi

NH2

CO O - P

carbamoyl-P

Carbamoyl-Psynthetase II (CPSII)C

Py1

Asp transcarbamoylaseAPy2

AspGln

CADPy 1-3

H2OA

6

AspGln

CADPy 1-3

H2OA““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis

3.DihydroorotaseD

Py3

NH2

C

O NH

carbamoyl-Asp

COO-

COO -

HN

O NH

Dihydroorotate (H2OA)

COO-

O

H2O

7

H2OAmito

Py 4OA ““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis

Dihydroorotatedehydrogenase

Py4

HN

O NH

orotate (OA)

COO-

O

4.NADH + H+

NAD+

8

UMP-SPy 5,6 OA

UMP

UMP-S(synthase)

Py5

Py6

““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis““de novo” PYRIMIDINE synthesisde novo” PYRIMIDINE synthesis

Orotatephosphoribosyl transferase 5.

PPi

PRPP

HN

O N COO-

O

R- POrotidylate (OMP)

UMP

6. CO2

OMP decarboxylase

9

UTP

UDP

UMP7.

Pyrimidine monophosphatekinase

ADP

ATPUMP

UDP

8.Pyrimidine diphosphate

kinaseADP

ATP

UTP (2,4-dioxo)

CTP

9.CTP synthetase

Glu

Gln

CTP (2-oxo-4 amino)

ADP

ATP

10

UMP

Regulation of Regulation of PYRIMIDINE “de novo” synthesisPYRIMIDINE “de novo” synthesis

Regulation of Regulation of PYRIMIDINE “de novo” synthesisPYRIMIDINE “de novo” synthesis

CADPy 1-3

CAD complex:a single polypeptidethree active centers+ allosteric sites

UTP

+ PRPP

UMP-SPy 5,6 OA

UMP

11

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

12

Oral uridine

UTP

UDP

UMP

CTP

AspGln

CADPy 1-3

H2OAmito

Py 4

UMP-SPy 5,6 OA

Orotic aciduriaOrotic aciduriaOrotic aciduriaOrotic aciduria

1. UMP-S deficiency

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

treatment: oral uridine

Main reasons of orotic aciduria

13

citidin

uridin

CMP

UMP

SALVAGESALVAGENucleoNucleosside ide nucleo nucleottideide

SALVAGESALVAGENucleoNucleosside ide nucleo nucleottideide

Cytidylate deaminase

UMP

Br -p

Br

uridine cytidine

CMP

Uridine/cytidine kinase

H2ONH3

ATP

ADP

ATP

ADP

14

Br -p

UMP (TMP) CMP

uridine (thymidine) cytidineB

r

Pi Pi

5’nucleotidase

cytidine deaminase

H2ONH3

CATABOLISMCATABOLISMNucleoNucleottide ide Nucleo Nucleossideide

CATABOLISMCATABOLISMNucleoNucleottide ide Nucleo Nucleossideidecytidine

uridine

CMP

UMP

15

cytidine deaminase

pyrimidine nucleoside phosphorylase

dihydropyrimidine dehydrogenase

dihydropyrimidinase

ureidopropionase

Coenzyme A

16

β-D-ribofuranose (in RNA) 2’-deoxy-β-D-ribofuranose (in DNA)

In cells, deoxyribonucleotides are synthesised from ribonucleotides by theribonucleotide reductase enzyme

Formation of deoxynucleotides

NADPH + H+ NADP+

H2O

17

The ribonucleotide reductase reaction The ribonucleotide reductase reaction (RR)(RR)

The ribonucleotide reductase reaction The ribonucleotide reductase reaction (RR)(RR)

RR

S

SRR

SH

SH

TR

S

STR

SH

SH

FADH2 FAD

NDP dNDP

TR:thioredoxin reductase

or:glutaredoxin reductase

(glutathion)

NDP:UDP/ADP/GDP/CDP

NADPH+ H+ NADP+

H2O

18

Mechanism of the RR reaction

ribonucleotide reductase

19

The RR is a dimer

R1 – catalytic subunit constitutive expression

R2 – regulatory subunit induced in the S-phase

RR is active in dividing cells!

20

Allosteric REGULATIONAllosteric REGULATIONAllosteric REGULATIONAllosteric REGULATION

1. dATP: complete inhibition

2. Regulation of the ratio

Substrates:

CDP

UDP

GDP

ADP

Products:

dCTP

dTTP

dGTP

dATP

+

+

dUDP dUMP

dCDP

21

Synthesis of thymine nucleotides

Folate antagonists(chemotherapy!)

dTTP DNA

UMP

UDP

dUDP

dUDP

22

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)

23

Inhibition of the folate synthesis: antibacterial effectInhibition of the folate synthesis: antibacterial effectInhibition of the folate synthesis: antibacterial effectInhibition of the folate synthesis: antibacterial effect

Human: folic acid is a vitaminBacteria: synthesis of folate can be selectively inhibited

H2N S

O

O

N

H

R

sulphonamides

folic acid

24

The role of folate and The role of folate and vitaminvitamin B B1212

in DNA synthesis and cell divisionin DNA synthesis and cell division

The role of folate and The role of folate and vitaminvitamin B B1212

in DNA synthesis and cell divisionin DNA synthesis and cell division

HomoCys

Met

THF

CH3-THF

CH2-THF

GlySer

dUMP dTMPpurine synthesis

Cell division

SAM

SAH-CH3

adenosine

B12

“methyl trap” B12/folate deficiency: pernicious anemia

oxidative stressatherosclerosis

folate cycleSAM/SAH

cycle

25

Folate deficiency – megaloblastic anemia

26

Vitamin B12 (cobalamin)deficiency

Methylmalonic aciduria Megaloblastic anemia(Pernicious anemia)

nerve degenerationparalysis and death

(B12 is needed for myelin synthesis)

Anemia is due to impaired dTTP synthesisfor DNA replication in hematopoetic precursor cells

might be due to achlorhydria (chronic gastritis, no IF production)treatment: parenteral administration of B12

27

“ “Salvage” reaction of Salvage” reaction of deoxydeoxynucleosidesnucleosides “ “Salvage” reaction of Salvage” reaction of deoxydeoxynucleosidesnucleosides

lymphoid specific

Deoxycytidine kinase (dCK)

Broad substrate specificity

dAdo, dGuo, dCyd

Analogues of deoxynucleotides: antileukemic agents

purine and pyrimidineanalogues

dCyd dCMP

ATP ADP

Substrates:

dThd dTMP

ATP ADP

Thymidine kinase (dTK)

28

Purine analogues

6-mercaptopurine 8-azaguanine2-chloro-2’-deoxyadenosine

(CdA, Cladribine®)

Hairy cell leukemia (HCL)

29

Pyrimidine analogues containing sugar derivatives

arabinosyl-cytosine(araC; for treating AML)

2’, 3’-dideoxy-cytidine 2’-deoxy-2’, 2’--difluorocytidine

(dFdC, Gemcitabine)

30

S-phase dependent enzyme

Thymidine kinase (dTK) dThd dTMP

ATP ADP

Human dTK: narrow substrate specificity

Herpes Simplex Virus : HSV- dTK: wide substrate specificity

Antiviral medication (Acyclovir)Suicide gene therapy (Gancyclovir, AIDS)

“ “Salvage” reaction of Salvage” reaction of deoxydeoxynucleosidesnucleosides “ “Salvage” reaction of Salvage” reaction of deoxydeoxynucleosidesnucleosides

31

Acyclovir Stavudine Didanosine

Antiviral nucleoside analogues

9-hydroxyethoxy-methyl guanine,

an acyclic nucleoside

inhibition of viral DNA (RNA) replicationherpes viruses, HIV

inhibitors of retroviralreverse transcriptase

32

„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

AspCO2, NH3 (Gln, Glu)

33

„salvage” pathway of purine- pyrimidine nucleotides

mainly from bases through PRPP reaction YES NO

PRPP is necessary YES NO

mainly from nucleosides NO YES

34

Catabolic pathway of purine- pyrimidine nucleotides

N of rings will be excreted YES NO

amino groups savedYES YES

diseases hyperuricemia orotic aciduria