PETER PAZMANY CATHOLIC UNIVERSITY

Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework**

Consortium leader

PETER PAZMANY CATHOLIC UNIVERSITY

Consortium members

SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER

The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund ***

**Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben

***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg.

BIOCHEMISTRY

Metabolism of nucleotides

(Nukleotidok metabolizmusa)

Raymund Machovich

Lecture objectives

To learn:

1) Importance of nucleotide synthesis

2) Synthesis of purine ring, formation of AMP and GMP 3) Significance of phosphoribosyl-pyrophosphate synthetase 4) Regulation of ATPand GTP synthesis

5) Salvage ways of purine nucleotide synthesis 6) Pyrimidine synthesis

7) Synthesis of deoxyribonucleotides and its control

8) Origins of deoxythymidilate: Thymidylate synthase, thymidine kinase 9) Some anticancer drugs

10) Degradation of purine bases – urate formation and its importance

Nucleotide structures

N N NH

N N

O

– P – O – CH

O O

O

OH OH

ester bond

Nucleotide: Adenosine –5'-monophosphate (AMP, adenylate, adenyl acid)

Nucleoside:

ribose (R) -base (B)

R B Adenosine ^DNS RNS

ATP cAMP NAD 180° ‘Anti”

„Syn”

N-glicoside bond

Diet

Resynthesis

Nucleotides

Biosynthetic Intermediers:

UDP-glucose

CDP-diacilglicerol

Redox systems Coenzymes

CoA, NAD, FAD

Regulation Adenosine ADP

cAMP cGMP

Energy ATP

Informations: RNA DNA

Various cells

DNS, RNS

Ribose – (R)

Deoxyribose (dR)- nucleases

Mono-(oligo-)nucleotides

Phosphatases

Nucleosides* Pi

Nucleosidases

Reuse R

dR Purine-

B _(d)R

Diet:

* Thymidine C

Uridine H

C

N C

C C

N

N

C N

1

2

3 4 5 6

7

8 9

Asp

CO

5

Gly

2

N

– Formyl-THF

3

Gln

4 1

N

– Formyl- THF

7

Synthesis of purine ring

P R ATP AMP

P R PP

IMP AMP GMP

Catalytic subunit

Regulator subunit

PRPP-synthetase

\

Purine ring is synthesized on ribose-phosphate

O

– P – O – CH

O O

O

OH OH

5

4

3 2

1

H

OH

O

– P – O – CH

O

O

OH OH H

O – P – O – P – O

ATP

AMP

PRPP - synthetase

H H

H O

H H H

═ ═

Ribose- 5 - phosphate

5–Phosphoribosyl–1–pyrophosphate (PRPP)

GMP IMP

= =

Gln Glu

PPi ^Azaszerin

(tumor chemotherapy)

O

– P – O – CH

O

O

O

OH OH NH

H H OH H

5 – Phosphoribosyl – 1 – amine

β ^COOH

C – NH₂ CH₂ O

C CH₂ N ═ N⁺ H

O ═

(Gln analog)

Gln-PRPP-amido-

transferase

NH₂

Ribose - P

ATP ADP Gly

Phosphoribosyl – glycinamide synthetase

Pi NH₂

CH₂ O C

NH

Ribose - P

Glycinamide ribonucleotide

N¹⁰– Formyl–THF Phosphoribozyl –glycinamide transferase

THF

Formylglycinamide ribonucleotide

H – C ═ O NH₂ CH₂ O ═C

NH

Ribose - P NH

CH₂ C ═ O

H HN ═ C

NH Ribose – P

ADP ATP

Glu Gln synthetase

Formylglycin –amidine ribonucleotide

ATP

Formylglycinamide ribonucleotide

Aminoimidazole

synthetase

ADP

HC C

H

N N N

CH

R ibose- P

ribonucleotide

HOOC

C

H

N N N

CH

Ribose- P

5 – Aminoimidazole – 4 – carboxy ribonucleotide

Asp ATP

synthetase CO₂

carboxylase

ADP

Succinylocarboxamide ribonucleotide

ADP

C

C

NH

N

N

CH

R ibose- P

HOOC – C – N – HOOC – CH

H

H ═O

Adenylosuccinate lyase

5 – Aminoimidazole– 4 – N –

succinylcarboxamide ribonucleotide

Aminoimidasole carboxamide

Fumarate

NH

N

N

CH

Ribóz- P

O═

C C

N N H

N

CH

Ribose- P

O═

C C

O ═ C H N^{10 –}Formyl – THF THF

Transferase

5 – Aminoimidazole – 4 – carboxamide–

ribonucleotide 5 – Formylaminoimidazole –

carboxamide – ribonucleotide H₂O

IMP – synthase

N N

CH

Ribose- P

O═

C C

HC

IMP – synthase

PRPP – kinase

}

Hypoxantine

Inozinate (IMP)

AMP GMP

N

N N N

CH

R - P

O═ C

C

HC

IMP

Adenylosuccinate synthetase

GDP GTP Pi

Asp

IMP dehydrogenase

NAD NADH H₂O

AMP GMP

Adenylosuccinate

Xanthylate

N N N

CH

R - P

N NH

C

C

HC

N N

N

CH

H R - P

O═ C

C

O ═ C HOOC – CH₂– C – COOH

H

Adenylosuccinate Xanthylate

Adenylosucccinate lyase Fumarate

N N N

CH

R - P

NH₂ C

C

HC

Adenylate AMP

N N N

=

CH

H R - P

O═ C

C

H₂N – C Gln

Glu ATP

AMP PPi

H₂O

GMP synthetase

Guanylate

GMP

ATP GTP

Gln analogous

=

Feedback controls of

biosynthesis of purine nucleotides

IMP

GTP

ATP

ATP ATP

ATP

Synthetase Dehydrogenase

Adenylo

succinate Xanthylate

PRPP

Ribose – 5 -P

PR-amine

Synthetase

Purine nucleotides can be re-used („Salvage ways”)

O - P – O – CH

O

H

O

H H OH OH

H

O – P – O – P - O^- O O

O^- O^-

=

PRPP

Adenine

Adenosine-phosphoribosyl transferase

PPi

HN C HC C

CH NH

N N N

CH

R - P

Adenylate

(AMP)

Ө

Hypoxantine + PRPP

phosphoribosyl transferase

PPi

Guanine + PRPP

PPi

HN C

HC C C O

N N N

CH

R - P

IMP

HN C C C

C N

CH O

H

N

AMP , GMP, IMP

inhibit their own syntheses

A – R

+ (G – R)

ATP ADP AMP

(GMP) Adenosine kinase

A: adenine

G: guanine

R: ribose

C

N C

C C

N

3

2

1 6 5 4

Pyrimidine syntheses

Carbamoyl phosphate Asp

H

N – C – O - P – O

O O

O

Glu 2ADP

Carbamoyl-phosphate synthetase (kDa 240)

Different carbamoylphosphate synthetases.

For pyrimidine: Gln in cytosol

For urea: NH

in mitochondria

H

N – C – O - P – O

O

H

N – C – COOH CH

COOH H

Asp

transcarbamoylase

Pi

HO – C ═ O NH₂

O ═ C CH₂ N C – COOH H H

N – carbamoyl-aspartate

CAD

Mr ≈ 240 000

Dihydroorotase Aspartate trans-

carbamylase

O C

HN CH

O ═ C C – COOH N H

H

═

Dihydroorotate

NAD

Dihydroorotate dehydrogenase

Dihydroorotase

H₂O

Orotate

C

HN CH

O ═ C C – COOH N

H

═

Orotate

═

Orotate aciduria

O C

HN CH

O ═ C C – COOH N

H

═

O^-- P – O – CH2

O

O^-

=

H

O H H OH OH PRPP PPi

Orotate-phosphorybosyl- transferase

Orotidylate

Transferase

Decarboxylase

Hereditary breakdown or azauridine (anticancer)

═

Orotidylate decarboxylase

decarboxylase

C

HN CH O ═ C C H

N Ribose – P

O C

HN CH O ═ C C H

N Ribose P

═

ADP ATP ADP ATP UDP

Nucleoside UMP- diphosphate kinase kináse

UTP

(uridine- triphosphate) ATP

ADP

Gln H₂O

CTP synthetase Glu

NH₂

C

N CH O ═ C CH

N

═

CTP

(cytidine triphosphate)

Uridylate UMP

Uridine monophosphate

Ribose –P – P – P

Deoxyribonucleotide synthesis

P - P – O – CH

H

O H H

OH

H

Base

P - P – O – CH

H

O H H

OH

H

NADPH ^NADP Base

H

O

5

4

3 2

1

Ribonucleotide reductase:

Its substrate: ribonucleotide diphosphate (BDP)

SH enzime:

SH

BDP + ε dBDP + H

O

S SH

ε

B

B

Regulation

ATP ⊕ dATP

SH SH

B

B

Fe ³⁺ - O - Fe ³⁺

Substrates:

ADP CDP UDP GDP

OH

O ·

\

\

dCDP dCTP

dUDP dTTP

dGDP dGTP CDP

UDP

GDP ATP

\ ⊕

⊕

⊕

Regulation of ribonucleotide reductase

„Uridine-way”

HN CH O = C CH

C

N

HN C – CH

C CH C

N O =

P dR P

N ⁵ N ¹⁰ - methylene - dR

THF

DHF

THF Gly

Ser

NADP

NADPH

Dihydrofolate reductase

dUMP

dTMP

Thymidylate synthase

HN C – CH

C CH C

N O

O = HN C – CH

C CH C

N O

O =

Deoxythimidylate (dTMP) synthesis:

„Thymidine-way”

H

O H H OH H

H

H

O

H H OH H

H

HO – CH

CH

Thymidine

kinase

ATP ADP

dT

(deoxythimidine)

dTMP

(Deoxythymidine-monophosphate)

DNA-degradation dT

H,

C

O^–

into RNA F- UDP, F - dUDP

Fluoro –deoxyuridylate (F – dUMP) O

C

HN C - F O ═ C C H

N P -dR

═

O C

HN C - F O ═ C C H

N

═ _THF

CH₂

S - enzym Thymidine

kinase dT

N,⁵N¹⁰Methylene THF

DHF Gly Ser

NADP

Dihydrofolate reductase NADPH

Compatitive inhibitors (K_i≈ 10^-9M)

Aminopterin Methotrexate

dTDP dTTP DNA

ATP ATP NTP

Pi Pi Polymerase

Kinases and phosphatases

Thymidilate - synthase

UDP

dUMP

dTMP

N

N

HO – CH

O

OH H H H OH

O═

Pyrimidine and purine analogues

N

N N H SH

N

6 –mercaptopurine („Immurán”)

Hypoxantine + PRPP

S – IMP Cytosine arabinose

═

Degradation of purines

N N N

CH

Ribose - P

N

NH₂ C

C

HC

N N N

CH

O C

C

HC

PRPP Gln

IMP

Adenylate deaminase

H₂O

Phosphomono

esterase

PRPP

Hypoxanthine phosphoribosyl transferase

- NH

AMP

Asp

Gln

Ribose Pi

Hypoxanthine

═

GMP

Xanthine

N N N

CH

O C

C

H₂N – C

═

N N H H

N

CH

O C

C

O ═ C

═

GMP

Phosphomonoesterase Ribose

Pi

Guanine

PRPP

H₂O

Guanine

deaminase

H

O

Xanthine oxidase H

O

Catalase

H

O

Xanthine

Guanine phosphoribsyl transferase

IMP

Hypoxanthine

N N H N

C –

HN OH C

C

HO– C

N N H H

N

C ═ O C

HN O C

C

O ═ C

═

H

O O

Xanthine oxidase

H

O

Catalase H

O

Uric acid (enol)

H

Urate (low solubility)

Gout,

Kidney stone

H

Uric acid (keto) Allantoin

urea

High level of urate results in deposition of sodium urate crystals

AMP

IMP

Hypo- xanthine

Xanthine

Guanine

Hypoxanthine – guanine- GMP

phosphoribozyl transferase

PRPP

ATP Ribose –5 –P

B PRPP-

synthetase

(regulator subunit defect)

Xanthine - oxidase

A

A

Genetic lack:

„Lesch Nyhan syndrome”

Bizarre reactions:

Self destructive tendencies (biting off their fingers, lip,

toes), bad coordination, mental retardardation

High level of uricacid results in its

Xanthine-

oxidase

Th: Allopurinol

Xanthine oxidase

Urate

joints kidney

N N H C C N N

OH C

C

H – C

N N

H N

C –H

OH C

C

H – C

H

Xanthine oxidases are inhibited (xanthine and hypoxanthine are more soluble than uric acid)

Allopurinol Hypoxanthine

Treatment of gout

Urate is decreased,

PRPP not

High urate level may be „useful”:

Urate level of the human is 10-fold higher than in monkey a factor for the longer life time.

Urate is an antioxaidant (like Vitamin E, bilirubin, etc).

Adenosine-deaminase

Its lack leads to a 100-fold increase in dATP, which inhibits ribonucleotide reductase.

The increase in dATP, thus results in decrease of other deoxinucleotides necessary for DNA synthesis

in T and B lymphocytes.

Its hereditary deficiency affects the immune system:

SCID-syndrome (sever combined immundeficiency).

It is also called „Non HIV AIDS”, because of similar symptoms.

Summary

Nucleotide metabolism is discussed from the viewpoint of their synthesis (because their amount is not enough in the diet) and they degradation (because they

incorrect elimination causes pathological conditions)

Recommended literature

Machovich R. A nucleotidok anyagcseréje

Orvosi Biokémia, Medicina kiadó,

pp. 269-296

What is the role of nucleotides?

1) Hydrolysis of PP bound of ATP to drive reactions that require an input of energy 2) Building blocks of DNA and RNA

3) Componenet of coenzymes 4) Second messengers

5) Allosteric effectors of metabolic procesess

Correct answere for test question:

„C”

Which of the compound(s) below is/are allosteric inhibitor(s) of PRPP amidotransferase?

1) ATP

2) IMP

3) PRPP

4) NADH

Correct answere for test question:

„C”