N

N

N

NH

N

N

Chapter 8

Metabolism of Nucleotides

The biochemistry and molecular biology department of CMU

Section 1 Introduction

Nucleoprotein

Nucleic acid Protein

Nucleotide

NucleosidePhosphate

Base Ribose

Nuclease

Nucleotidase

Nucleosidase

Degradation of nucleic acid

Significances of nucleotides

1. Precursors for DNA and RNA synthesis

2. Essential carriers of chemical energy, especially ATP

3. Components of the cofactors NAD+, FAD, and coenzyme A

Significances of nucleotides (continue)

4. Formation of activated intermediates such as UDP-glucose and CDP-diacylglycerol.

5. cAMP and cGMP, are also cellular second messengers.

There are two pathways leading to nucleotides

De novo synthesis: The synthesis of nucleotides begins with their metabolic precursors: amino acids, ribose-5-phosphate, CO2, and one-carbon units.

Salvage pathways: The synthesis of nucleotide by recycle the free bases or nucleosides released from nucleic acid breakdown.

Section 2 Synthesis of Purine Nucleotides

§ 2.1 De novo synthesis• Site: in cytosol of liver, small intestin

e and thymus

• Characteristics:

a. Purines are synthesized using 5-phosphoribose as the starting material step by step.

b. PRPP is active donor of R-5-P.

c. AMP and GMP are synthesized further at the base of IMP.

1. Element sources of purine bases

N

CN

C

CC

N

C

N

CO2

One carbon unit

Gln

Gly

Asp

1

23

45 7

89

6One carbon unit

2. Synthesis of IMP

HN

N

N

N

R-5-P PRPP

ATP AMP

PRPPkinase

Gln Glu

amidotransferase5-phospho-

ribosylamine(PRA)

9 stepsO

R- 5'-P

( IMP )inosine 5-monophosphate

Glnamidotransferase

O

OH

H H

OH

H

H

CH2O

O PP O

P

O

OH

H H

OH

OH

H

H

CH2OPAMPATP

Mg2+

5-phosphoribose1-pyrophosphate

£¨PRPP£©

5-phosphoribose(R-5-P)

PRPPkinase

PPi

O

OH

H H

OH

H

NH2

H

CH2OP

5-phosphoribosyl-amine (PRA)

Glu

HO

H2NC

CC

N

CH

N

O

R-5'-PH2N

C

HC

N

CH

N

R-5'-P

C

H2C

NH

CH

HN

R-5'-P

HNO

C

H2C

NH

CH

HN

R-5'-P

OO

CO2

H2O

ATP

O

OH

H H

OH

H

HN

H

CH2P OCO

H2C

NH2

OHCO

H2CNH2

FH4

ATP

O

OH

H H

OH

H

NH2

H

CH2OP

Carboxyamino-imidazole

ribonucleotide£¨CAIR£©

5-aminoimidazoleribonucleotide

£¨AIR£©

Formylglycinamidineribonucleotide

£¨FGAM£©

Formylglycinamideribonucleotide

£¨FGAR£©

carboxylase

Glycinamideribonucleotide

£¨GAR£©

Gly

transformylase

ADP+Pi

GARsynthetase

PRA

N10-CHO FH4

FGAM synthetase

AIRsynthetase

ATP

Gln

Glu

HN

HCN

C

CC

N

CH

N

O

R-5'-P

H2N

HC

NH

C

CC

N

CH

N

O

R-5'-P

O

H2N

H2NC

CC

N

CH

N

O

R-5'-P

H2O

FH4

NH

H2NC

CC

N

CH

N

O

R-5'-P

HC

CH2

COOH

COOH

HO

H2NC

CC

N

CH

N

O

R-5'-P

H2OATP

IMP 5-formaminoimidazole- 4-carboxamide £¨FAICAR£©

5-aminoimidazole-4-carboxamide ribonucleotide

£¨AICAR£©

cyclohydrolase

transformylase

N10-CHO FH4

5-aminoimidazole-4-(N-succinylcarboxamide)

ribonucleotide (SAICAR)

Fumarate

lyase

Carboxyamino-imidazole

ribonucleotide£¨CAIR£©

Asp

synthetase

N10-CHOFH4

N10-CHOFH4

3. Synthesis of AMP and GMP

IMP

XMP

H2O

ATP

Gln Glu

Adenylsuccinate £¨ AMPS£©

AMPS lyase

Fumarate

GMP synthetase

NAD+ + H2O

HN

N

N

N

NH

R-5'-P

HN

NH

N

N

O

R-5'-P

CHHOOC CH2 COOH

O

HN

NH

N

N

O

R-5'-P

AMP

HN

N

N

N

NH2

R-5'-P

GMP

HN

N

N

NH2N

O

R-5'-P

GTPAsp

NADH + H+

AMPSsynthetase

IMPdehydrogenase

4. Formation of NDP and NTP

AMP + ATP 　　　 2 ADP

NDP + ATP 　　　 NTP + ADP

Adenylate kinase

nucleoside diphosphate kinase

5. Regulation of de novo synthesis

The significance of regulation:

(1) Fulfill the need of the body, without wasting.

(2) [GTP]=[ATP]

R-5-P

ATPPRPP PRA IMP

AMPS

XMP

AMP

GMP

ADP

GDP GTP

ATP

＋

＋＋

＋

－ －

－ － －

－ －

－

Regulation of de novo synthesis of purine nucleotides

§ 2.2 Salvage pathway

The significance of salvage pathway :

a. Save the fuel.

b. Some tissues and organs such as brain and bone marrow are only capable of synthesizing nucleotides by a salvage pathway.

The course of salvage pathway :

Hypoxanthine

IMP

HGPRT

PRPP

PPi

Guanine

GMP

PPi

APRT

PRPP

Adenine

AMP

Hypoxanthine

IMP

HGPRT

PRPP

PPi

Hypoxanthine

IMP

HGPRT

PRPP

PPi

Guanine

GMP

PPi

Guanine

GMP

PPi

APRT

PRPP

Adenine

AMP

APRT

PRPP

Adenine

AMP

Adenine

AMP

- -

adenosine AMP

ATP ADP

adenylate kinase

• HGPRT: Hypoxanthine-guanine phosphoribosyl transferase

• APRT: Adenine phosphoribosyl transferase

• Absence of activity of HGPRT leads to Lesch-Nyhan syndrome.

§ 2.3 Exchange between purines

AMP

AMPS IMP XMP

GMPH2O

NH3NADP+

NH3

NADPH + H+

adenosine

deaminaseguanosin

e

reducta

se

§ 2. 4 Formation of deoxyribonucleotide

• Formation of deoxyribonucleotide involves the reduction of the sugar moiety of ribonucleoside diphosphates (ADP, GDP, CDP or UDP).

• Deoxyribonucleotide synthesis at the nucleoside diphosphate level.

SS

H2OMg2+

NADPH + H+NADP+

SHSH

thioredoxin

ribonucleotide reductase

NDP£¨N=A, G, C, U£©

dNDP

dNTP

ATP

ADP

kinase

O BaseCH2

HOH

OP PO BaseCH2

OHOH

OP P

thioredoxin

thioredoxin reductase

FAD

Regulation of ribonucleotide reductase

CDP dCDP dCTP

ATP

UDP dUDP dTTP

GDP dGDP dGTP

ADP dADP dATP

§ 2. 5 Antimetabolites of purine nucleotides

• Antimetabolites of purine nucleotides are structural analogs of purine, amino acids and folic acid. They can interfere, inhibit or block synthesis pathway of purine nucleotides and further block synthesis of RNA, DNA, and proteins.

1. Purine analogs

• 6-Mercaptopurine (6-MP) is a analog of hypoxanthine.

N

N NH

N

OH

N

N NH

N

SH

6-MPhypoxanthine

6-MP 6-MP nucleotide

de novo synthesis

salvage pathway

HGPRT

amidotransferase

IMP

AMP and GMP

--

-

-

-

• 6-MP nucleotide is a analog of IMP

2. Amino acid analogs

• Azaserine (AS) is a analog of Gln.

H2N C CH2

O

CH2 CH

NH2

COOH Gln

C

O

CH2 CH

NH2

COOH ASNN CH2 O

3. Folic acid analogs

• Aminopterin (AP) and Methotrexate (MTX)

R＝H：AP

folic acid

N

NN

N

NH2

H2N

CH2 N C

R O

NH CH

COOH

R＝CH3：TXT

CH2 CH2 COOH

N

NN

N

OH

H2N

CH2 N C

H O

NH CH

COOH

CH2 CH2 COOH

folate FH2 FH4

NADPH + H+

NADP+NADPH + H+

NADP+

FH2 reductase FH2 reductase

AP or MTX

- -

Section 3 Catabolism of Purine Nucleotides

nucleotide

nucleotidaseH2O

Pi

nucleosidenucleoside

phosphorylase

Pi R-1-P

purine

R-5-P

PRPP

pentose phosphate pathway

salvage pathway

oxidationuric acid

N

HCN

C

CC

N

CH

N

NH2

Ribose-P

AMP

HN

HCN

C

CC

N

CH

N

O

Ribose-P

IMPHN

HCN

C

CC

NH

CH

N

O

HN

CNH

C

CC

NH

CH

N

O

O

HN

CNH

C

CC

NH

C

N

O

O

O

GMP

Hypoxanthine

Uric Acid Xanthine

Xanthine Oxidase

• Uric acid is the excreted end product of purine catabolism in primates, birds, and some other animals.

• The rate of uric acid excretion by the normal adult human is about 0.6 g/24 h, arising in part from ingested purines and in part from the turnover of the purine nucleotides of nucleic acids.

• The disease gout, is a disease of the joints, usually in males, caused by an elevated concentration of uric acid in the blood and tissues. The joints become inflamed, painful, and arthritic, owing to the abnormal deposition of crystals of sodium urate. The kidneys are also affected, because excess uric acid is deposited in the kidney tubules.

HN

HCN

C

CC

NH

CH

N

O

Hypoxanthine

HN

HCN

C

CC

NH

N

HC

O

Allopurinol

Allopurinol – a suicide inhibitor used to treat Gout

Section 4 Synthesis of P

yrimidine Nucleotides

§ 4.1 De novo synthesisCharacteristics:

• The enzymes mostly lie in cytosol, but some enzymes exist in mitochondria.

• The pyrimidine ring is first synthesized, then combines with PRPP.

• UMP is first synthesized, then UMP is used for synthesizing other pyrimidine nucleotides.

1. Element source of pyrimidine base

N

CN

C

CC

12

34

5

6Asp

CO2

Gln

2. Synthesis of UMP

Carbamoyl phosphate synthetase ¢ò

2ATP 2ADP+Pi

+ GluGln HCO3-+

(CPS-II)Carbamoyl phosphate

C OPO3H2

H2N

O

CPSⅠ CPSⅡ

location Mit （ liver) Cytosol (all the c

ell)

source of nitrogen

NH3 Gln

allosteric agent AGA none

function urea

synthesis pyrimidine biosy

nthesis

Difference of carbamoyl phosphate synthetase and Ⅰ Ⅱ

HN

N

O

O

HN

N

O

O COOH

HN

NH

O

O COOH

HN

CNH

C

CH2

C

O

O COOHH

NH2

CNH

C

CH2

C

O

O COOHH

HONH2

C

H2NC

CH2

C

OO

COOHH

HO

O P

carbamoylphosphate

carbamoylaspartate

H2O

dihydroorotase

dihydroorotateNAD+

NADH+H+

orotic acid

PRPPPPi

orotidinemonophosphate £¨ OMP£©

CO2

OMP decarboxylase

UMP

Pi

R-5'-P

orotatephosphoribosyl transferase

R-5'-P

Aspartatetranscarbamoylase

Aspdihydroorotatedehydrogenase

UTP

3. Synthesis of CTP

Amidation at the nucleoside triphosphate level.

UTP CTP

Gln + ATP

Glu + ADP + Pi

CTP synthetase

N

N

NH2

O

R 5' PPP

HN

N

O

O

R 5' PPP

4. Formation of dTMP

The immediate precursor of thymidylate (dTMP) is dUMP.

The formation of dUMP either by deamination of dCMP or by hydrolyzation of dUDP. The former is the main route.

dTMP synthesis at the nucleoside monophosphate level.

dUMP

dUDP

dCMPdTMP

H2O

Pi

H2O

NH3

NADPH

NADP+

thymidylate synthase HN

N

O

O

R 5' Pd

CH3

reductase

HN

N

O

O

R 5' Pd

+ H+

FH2

FH4

N5, N10-CH2-FH4 FH2

5. Regulation of de novo synthesis

carbamoyl phosphate

carbamoyl aspartate

UMP

ATP + CO2 + Gln

PRPP

UTP CTP

ATP + R-5-P

purine nucleotide

pyrimidine nucleotide

§ 4. 2 Salvage pathway

+ ATP

+ ATP

+ ATP

UMPCMP

dTMP + ADP

dCMP + ADP

uridinecytidine

deoxythymidine

deoxycytidine

thymidine kinase

deoxycytidine kinase

uridine-cytidine kinase+ ADP

+ PRPP + PPiuracilthymineorotic acid

pyrimidine phosphate ribosyltransferase UMP

dTMPOMP

§ 4. 3 Antimetabolites of pyrimidine nucleotides

• Antimetabolites of pyrimidine nucleotides are similar with them of purine nucleotides.

1. Pyrimidine analogs

• 5-fluorouracil (5-FU) is a analog of thymine.

HN

NH

O

O

FHN

NH

O

O

CH3

thymine5-FU

5-FU5-FdUMP

5-FUTP

dUMP dTMP

Synthesis of RNA

Destroy structure of RNA

2. Amino acid analogs

• AS inhibits the synthesis of CTP.

3. Folic acid analogs

• MTX inhibits the synthesis of dTMP.

4. Nucleoside analogs

• Arabinosyl cytosine (ara-c) inhibits the synthesis of dCDP.

N

N

NH2

O

ara-c

O

H

OH H

H

CH2OH

H OH

N

N

NH2

O

cytosine

O

H

OH OH

H

CH2OH

H H

Section 5 Catabolism of

Pyrimidine Nucleotides

+

CO2, NH3

H2OH2O

H2N CH2 CH2 COOH H2N CH2 CH COOH

CH3

N

NH

O

NH2H2O NH3

HN

NH

O

O

CH2

CH2NH2

NH

O

HOOC

HN

NH

O

O

CH3

CH2

CHNH2

NH

O

HOOC

CH3

cytosine uracil thymine

¦Â-ureidopropionate

¦Â-ureido-isobutyrate

CO2 + NH3

¦Â-alanine ¦Â-aminoisobutyrate

Summary of purine biosynthesis

　dATP

dGTP

AMP

GMP

ADP

GDP

dADP

dGDP

IMP

ATP

GTP

CTP

Summary of pyrimidine biosynthesis

　

UDP UTP

CDP

dUDP

dCDP

dUMP

dCMP

dTMP

UMP

dTDP dTTP

dCTP