chapter 8. nucleotide metabolism

Chapter 8. Nucleotide Metabolism

Functions of nucleotites:They are precursors of DNA and RNAATP is a universal currency of energyPhysiological mediators (cAMP, cGMP)They are activated intermediates in many biosynthesis (UDP-glucose, CDP-choline)Adenine nucleotides are components of some coenzymes (NAD+, NADP+ and FAD )

1. Biosynthesis of purine nucleotites:

1) The origins of the atoms in the purine ring:

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Glutamine

N10-Formyl-tetrahydrofolate

CO2

Aspartate

N10-Formyl-tetrahydrofolate

Glycine

N

CN

C

CC

N

C

N

One C unit

One C unit

2) Formation of phosphoribosylamine

Ribose 5-phosphate PRPPPRPP synthase ADPATP

PRPP: 5-Phosphoribose-1-pyrophosphate

OCH2

HH

OH

OP

O-O

O-H

OH

OH

H OCH2

HH

OH

OP

O-O

O-H

OH

O

H

P O P O-O

O-O-

OATP AMP

PRPP synthase

PRPP 5-phosphoribosyl-1-amineGln PRPP

Amidotransferase

Glutamine glutamate + PPi

OCH2

HH

OH

OP

O-O

O-H

OH

O

H

P O P O-O

O-O-

O OCH2

HH

OH

OP

O-O

O-H

OH

H

NH2

Aminophosphoribosyltransferase

Gln Glu + PPi

3) Formation of inosinate (IMP)

OCH2

HH

OH

OP

O-O

O-H

OH

H

NH2

Ribose-P

N

CHN

C

CN

CO

HN

CH

Ribose -P

NH

C

CH2

NH

HN

CO H

Ribose-PN

CHN

HCCH2N

Ribo se -P

NH

C

CH2

NH

O

CHO

Ribose-P

N

CHN

CCH2N

-OOC

Ribose-P

N

CHN

CCH2N

C

O

NHCH-OOC

CH2

COO-

Ribose-P

N

CHN

C

CH2N

C

O

H2N

Ribose-P

N

CHN

CCN

CO

H2N

COH

Ribose-P

NH

C

CH2

NH 3+

OGly,ATP

N10-formylFH4

Gln,ATP -H2O

CO2

Asp,ATPFumarate

N10-formylFH4

-H2O

(IMP)

4) Formation of AMP and GMP

(IMP)Ribose-P

N

CHN

C

CN

CO

HN

CH

Ribose-P

N

CHN

C

CN

CNH

N

CH

CH COO-CH2-OOC

Ribose-P

N

CHN

C

CN

C

NH2

N

CH

Ribose-P

N

CHN

C

CN

CO

HN

CO

H Ribose-P

N

CHN

C

CN

CO

HN

CH2N

Asp,GTP

Fumarate

H2O,NAD+

Gln,ATP

(AMP)

(GMP)

5) Formation of ATP and GTP

AMP ADP ATP

GMP GDP GTP

Kinase

Kinase Kinase

Phosphorylation

ATP ADP

ATP ADPATP ADP

Pi

6) Regulation of purine nucleotite synthesisFeedback inhibitors: AMP, ADP, GMP, GDP, IMP

AMP ADP ATPR-5-P PRPP PRA IMP GMP GDP GTP

PRA: 5-phosphoribosyl-1-amine . activation inhibition

7) Salvage pathways for purine nucleotite synthesis

PRPP Purine ribonucleotide

Adenine + PRPP AMP + PPiHypoxanthine + PRPP IMP + PPiGuanine + PRPP GMP + PPiAdenosine + ATP AMP +ADP

OCH2

HH

OH

OP

O-O

O-H

OH

O

H

P O P O-O

O-O-

OOCH2

HH

OH

OP

O-O

O-H

OH

H

PurinePurine PPi

8) Biosynthesis of deoxyribonucleotides(At the NDP level)

Ribonucleoside deoxyribonucleoside diphosphate diphosphate

Base=purine or pyrimidine

OCH2

HH

OH

OP

O

O

O-H

OH

H

BaseP

O

O-

-O OCH2

HH

OH

OP

O

O

O-H

H

H

BaseP

O

O-

-ONADPH+H+ NADP+ + H2O

Ribonucleotide reductase

The mechanism for ribonucleotide reduction:NDP dNDP

Ribonucleotide SHreductase SH

FADH2

Ribonucleotide Sreductase S

Thioredoxin SH SH

Thioredoxin S S

FAD

NADP+ NADPH + H+

Thioredoxinreductase

8) Inhibition of purine nucleotide biosynthesis by some anticancer drugs

Reaction InhibitorPRPP PRA 6-mercaptopurine(6MP)Glycinamide ribonucleotide Methotrexate(MTX)Formylglycinamide ribonucleotide Formylglycinamide ribonucleotide AzaserineFormylglycinamidine ribonucleotideIMP AMP 6MPIMP GMP 6MP, AzaserineAdenine AMP 6MPGuanine GMP 6MP

6MP

N

CHN

C

CN

CSH

N

CH

H

+N N CH2 C

O

O CH2 CH

NH2

COO-

Azaserine

N

CN

C

CC

NCH

CN

NH2

H2N

CH2 N

CH3

C

O

NH CH

COO-

CH2 CH2 COO-

Methotrexate (MTX)

2. Degradation of purine nucleotides:

AMP IMP Hypoxanthine Xanthine Uric acidGMP Guanine Xanthine Uric acid

N

CN

C

CN

CO

HN

CO

H H

H

O

N

CN

C

CN

COH

N

CHO

H

O-

Uric acid Urate

N

CHN

C

CN

CO

HN

CO

H HN

CHN

C

CN

CO

HN

CH

H

N

CN

C

CN

CO

HN

CO

H H

H

O

Ribose-P

N

CHN

C

CN

CO

HN

CH

IMP Hypoxanthine Xanthine Uric acid

Ribose-P

N

CHN

C

CN

CO

HN

CH2NN

CHN

C

CN

CO

HN

CH2N

H

N

CHN

C

CN

CO

HN

CO

H H

GMP XanthineGuanine

Ribose-P

N

CHN

C

CN

CO

HN

CH

Ribose-P

N

CHN

C

CN

CNH2

N

CH

AMP IMP

Gout is induced by high serum levels of urate, a disease that affects the joints and kidneys.

Allopurinol is an analog of hypoxanthine. It is extensively used to treat gout.

In the body, allopurinol is converted into alloxanthine, which then remains tightly bound to the active site of xanthine oxidase and thus inhibits the production of urate.

N

NC

C

CN

COH

N

CH

H

H

N

NC

C

CN

COH

N

CHO

H

H

Allopurinol Alloxanthine

Xanthine oxidase

3. Synthesis of pyrimidine nucleotides:

1) Origins of the atoms in the pyrimidine ring

C N C

C C N

Carbamoylphosphate

Aspartate

2) Pathway of pyrimidine nucleotide synthesisA) Formation of carbamoyl phosphate

2ATP + HCO3- + 2ADP+ PiH2N C

O

O P O-O

O-

Gln Glu

Differences between carbamoyl phosphate biosynthesis in the pyrimidine pathway and that in the urea cycle

In pyrimidine pathway In urea cycleLocation Cytosol Mitochondria-NH2 from: Gln NH4

+

Enzyme Carbamoyl phosphate Carbamoyl phosphate synthase-II synthase-IN-acetyl-Glu No effect Activator

B) Formation of orotate

H2N C

O

O P O-O

O-+ H2N C

COO-

H

CH2

COO-

Asp transcarbamoylase

PiH2N C

O

HN C

COO-

H

CH2

COO-

Carbamoyl phosphate Asp N-Carbamoylaspartate

Dihydroorotase

CHN

CN

CH

CH2

O

O

H

COO-

H+

H2O

Dihydroorotate

Dihydroorotatedehydrogenase

NADH + H+ NAD+C

HN

CN

C

CH

O

O

H

COO-

Orotate

C) Formation of UMP

CHN

CN

C

CH

O

O

H

COO-

PRPP PPi

OCH2

HH H

OHOHH

OP-O

O

O-

CHN

CN

CCH

O

OCOO- H+ CO2

OCH2

HH H

OHOHH

OP-OO

O-

CHN

CN

CHCH

O

OOrotatephosphoribosyl

transferase

Orotidylatedecarboxylase

Orotate Orotidylate UMP

C) Formation of other pyrimidine nucleotides

UMP UDP UTPATP ADPATP ADP

Kinase Kinase

UTP

OPOPHOO O

O- O-OCH2

HH H

OHOHH

OPO

O-

CHN

CN

CHCH

O

OOPOPHO

O O

O- O-OCH2

HH H

OHOHH

OPO

O-

CNC

NCHCH

NH2

O

CTP

Gln, ATP Glu, ADP H2O Pi, 2H+

CTP synthase

UDP dUDP dUTP

dUTP dUMP

dUMP

OCH2

HH H

HOHH

OP-OO

O-

CHN

CN

CHCH

O

O

OCH2

HH H

HOHH

OP-OO

O-

CHN

CN

CHC

O

O

CH3

dTMP

Thymidylate synthase

N5N10-MethyleneFH4

FH2

NADPH NADP+, H2O

Ribonucleotidereductase

ATP ADP

Kinase

H2O PPi

dUTPase

D) Regulation of pyrimidine nucleotide synthesis

CO2 + Gln + ATP

Carbamoyl phosphate

N-Carbamoylaspartate

UMP

Inhibited by UMP

Inhibited by CTP

-

-

3) Salvage pathway for pyrimidine nucleotide biosynthesis

Pyrimidine Pyrimidine ribonucleoside monophosphate

Orotate Orotidylate UMP

PRPP PPi

PyrimidinePhosphoribosyltransferase

PRPP PPi

OrotatePhosphoribosyltransferase

H+ CO2

Orotidylatedecarboxylase

4) Anticancer drugs that block the pyrimidine nucleotide biosynthesis

A) 5-Fluorouracil (5-FU): is converted in vivo into fluorodeoxyuridylate (F-dUMP), which is an analog of dUMP and irreversibly inhibits thymidylate synthase.

F-dUMP

OCH2

HH H

HOHH

OP-OO

O-

CHN

CN

CHC

O

O

F

OCH2

HH H

HOHH

OP-OO

O-

CHN

CN

CHC

O-

O

F

S Enzyme OCH2

HH H

HOHH

OP-OO

O-

CHN

CN

CHC

O

OF

S Enzyme

CH2 FH4

HS-E Methylene FH4

B) Aminopterin and methotrexate (MTX): both are analogs of dihydrofolate. They are potent competitive inhibitors of dihydrofolate reductase and thus block the reactions using one-carbon units.

N

N

N

NH2N

NH2

CH2

H

NH C

O

NH CHCOO-

CH2 CH2 COO-

Aminopterin

C) Cytosine arabinoside and azaserine: Cytosine arabinoside is an analog of cytosine riboside and thus inhibits the reduction of CDP to dCDP, while azaserine inhibits the conversion of UTP to CTP.

OCH2

HH HO

HOHH

HO

CNC

NCHCH

NH2

O

Cytosine arabinoside

UMP UTP CTP CDP dCDP

UDP dUDP dUMP dTMP

Azaserine Cytosine arabinoside

5-FU

Aminopterin

-

-

-

-

4. Degradation of pyrimidine nucleotide The pyrimidine ring can be completely de

graded in humans. The products include: NH3, CO2, -alanine, and -aminoisobutyrate. Both -alanine, and -aminoisobutyrate can be further converted into acetyl-CoA and succinyl-CoA, respectively, or are excreted in the urine.

CMPHO OCH2

HH H

OHOHH

CHN

CN

CHCH

O

OHO OCH2

HH H

OHOHH

CNC

NCHCH

NH2

OH2O Pi H2O NH3

UMP

OCH2

HH H

OHOHH

OP-OO

O-

CHN

CN

CHCH

O

O

Cytidine Uridine

Pi H2O

CHN

CN

CHCH

O

OH

Pi Ribose 1-phosphate

UracilDihydrouracil

CHN

CN

CH2

CH2

O

OH

NADP+ NADPHC

CN

CH2

CH2

O

OH

H3N+

-O

-Ureidopropionate

H2OC

H3N+ CH2

CH2

O-O

-Alanine

CO2 +NH3 H2O

C

CHOCH2

O-O

Malonatesemialdehyde

COCH3

SCoA

Acetyl-CoA

NAD+ NADHHSCoA CO2

Pyruvate

Alanine

dCMPO OCH2

HH H

HOHH

CHN

CN

CHC

O

O

CH3

PO

-O

O-

H2O NH3 FH4=CH2 FH2

dUMP

OCH2

HH H

HOHH

OP-OO

O-

CHN

CN

CHCH

O

O

dTMP

CHN

CN

CHC

O

OH

CH3

Pi Ribose 1-phosphate

ThymineDihydrothymine

CHN

CN

CH2

CH

O

OH

CH3NADP+ NADPHC

CN

CH2

CH

O

OH

H3N+

-OCH3

-Ureido- -methylpropionate

H2OC

H3N+ CH2

CH

O-O

CH3

-Amino-isobutyrate

CO2 +NH3 H2O

C

CHOCH

O-O

CH3

Methylmalonatesemialdehyde

COCH2

SCoA

CH3

Propionyl-CoA

NAD+ NADHHSCoA CO2

Pyruvate

Alanine

OCH2

HH H

HOHH

CHN

CN

CHC

O

O

CH3

HO

Deoxythymidine

H2O Pi

COCH2

SCoA

CH2 COO-

Succinyl-CoA

5. Dysmetabolism of nucleotides Disease Deficiency SymptomsGout PRPP synthetase hyper uricemiaLesch-Nyhan HGPRT purine and uricsyndrom acid, cerebral

paralysisImmunodeficiency adenosine deaminase deoxyadenosineDiseases uria, dysostosisKidney stone APRT kidney stone,

urodynia, hematuriaXanthinuria xanthine oxidase kidney stone,

no symptoms