Seminar 11

Structure of nucleotides

Nitrogenous base + ribose

Nitrogenous base + ribose + phosphate

Nucleoside =

Nucleotide =

Nucleoside and Nucleotide

Purines vs Pyrimidines

pyrimidine purine OR

(Ribose or 2-deoxyribose)

N-b-glycosyl bond

Structure of nucleotides

Degradation of nucleic acids

Nucleoprotein

Nucleic acid Protein

Nucleotide

Nucleoside Phosphate

Base Ribose

Nucleotidase

Nucleosidase

In stomach Gastric acid and pepsin

In small intestine Endonucleases: RNase and DNase

Degradation of nucleic acid

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

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

5. cAMP and cGMP, are also cellular second messengers

Significances of nucleotides

Synthesis of of purine nucleotides

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

• Salvage pathways

The synthesis of nucleotide by recycle the free bases or

nucleosides released from nucleic acid breakdown

Synthesis of Purine Nucleotides

Site

• in cytosol of liver, small intestine and thymus

Characteristics

• Purines are synthesized using 5-phosphoribose (R-5-P)

as the starting material step by step

• PRPP (5-phosphoribosyl-1-pyrophosphate) is active

donor of R-5-P

• AMP and GMP are synthesized further at the base of

IMP (Inosine-5-Monophosphate)

De novo synthesis

N10-Formyltetrahydrofolate

N10-Formyltetrahydrofolate

Element sources of purine bases

FH4 (or THF)

N10—CHO—FH4

Important compounds

N10-Formyltetrahydrofolate

CHO

• basic pathway for biosynthesis of purine

ribonucleotides

• starts from ribose-5-phosphate (R-5-P)

• Requires 11 steps overall

• occurs primarily in the liver

Synthesis of Inosine Monophosphate (IMP)

OH

1 ATP

AMP

2

Gln:PRPP amidotransferase

ribose phosphate pyrophosphokinase / PRPP synthetase

Step 1: Activation of ribose-5-phosphate

Step 2: Acquisition of purine atom N9

(PRA)

Steps 1 and 2 are tightly regulated by feedback inhibition

3

Step 3: Acquisition of purine atoms C4, C5, and N7

glycinamide synthetase

4

Step 4: Acquisition of purine atom C8

formyltrasferase

5

Step 5: Acquisition of purine atom N3

synthetase

6

Step 6: Closing of the imidazole ring

synthetase

(CAIR)

7

Step 7: Acquisition of C6

AIR carboxylase

Step 8: Acquisition of N1

SAICAR synthetase

Step 9: Elimination of fumarate

adenylosuccinate lyase

Step 10: acquisition of C2

formyltransferase

Step 11: Ring closure to form IMP

Once formed, IMP is rapidly converted to AMP and GMP (it does not accumulate in cells)

Note: GTP is used for AMP synthesis

Note: ATP is used for GMP synthesis

IMP is the precursor for both AMP and GMP

Conversion of IMP to AMP and GMP

kinase

ADP

kinase

ADP

ATP

ATP ADP

AMP

ATP

kinase

GDP

kinase

ADP

GTP

ATP ADP

GMP

ATP

ADP, ATP, GDP and GTP biosynthesis

The significance of regulation:

1. Meet the need of the body, without wasting

2. AMP and GMP control their respective synthesis from IMP by a feedback mechanism [GTP]=[ATP]

Regulation of de novo synthesis

Purine nucleotide biosynthesis is regulated by feedback inhibition

• Purine bases created by degradation of RNA or DNA and

intermediate of purine synthesis can be directly converted to the

corresponding nucleotides

• The significance of salvage pathway

– save the fuel

– some tissues and organs such as brain and bone marrow are

only capable of synthesizing nucleotides by salvage pathway

• Two phosphoribosyl transferases are involved

– APRT (adenine phosphoribosyl transferase) for adenine

– HGPRT (hypoxanthine guanine phosphoribosyl transferase)

for guanine or hypoxanthine

Salvage pathway

Purine Salvage Pathway

N

NN

N

NH2

O

Guanine

N

N N

O

N

Hypoxanthine

O

OHHO

2-O3POH2C

N

N N

O

N

IMP

O

OHHO

2-O3POH2C

N

NN

N

NH2

O

GMP

.

.

Adenine AMP

PRPP PPi

adenine phosphoribosyl transferase

PRPP PPi

hypoxanthine-guaninephosphoribosyl transferase

(HGPRT)

Absence of activity of HGPRT leads to Lesch-Nyhan syndrome

Lesch-Nyhan syndrome

• first described in 1964 by Michael Lesch and William L. Nyhan

• there is a defect or lack in the HGPRT enzyme

• sex-linked metabolic disorder: only males

• the rate of purine synthesis is increased about 200-fold

– loss of HGPRT leads to elevated PRPP levels and stimulation of de novo

purine synthesis

• uric acid level rises and there is gout

• in addition there are mental aberrations

• patients will self-mutilate by biting lips and fingers off

Lesch-Nyhan syndrome

• formation of deoxyribonucleotide involves the

reduction of the sugar moiety of ribonucleoside

diphosphates (ADP, GDP, CDP or UDP)

• deoxyribonucleotide synthesis at the nucleoside

diphosphate(NDP) level

Formation of deoxyribonucleotide

S

S

H2OMg

2+

NADPH + H+

NADP+

SH

SHthioredoxin

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

Deoxyribonucleotide synthesis at the NDP level

• 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 DNA, RNA, and proteins

• widely used to control cancer

Antimetabolites of purine nucleotides

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

N

N NH

N

OH

N

N NH

N

SH

6-MPhypoxanthine

Purine analogs

6-MP 6-MP nucleotide

de novo synthesis

salvage pathway

HGPRT

amidotransferase

IMP

AMP and GMP

-

-

-

-

-

6-MP nucleotide is a analog of IMP

• Azaserine (AS) is a analog of Gln (glutamine)

H2N C CH2

O

CH2 CH

NH2

COOH Gln

C

O

CH2 CH

NH2

COOH ASNN CH2 O

Amino 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

MTX

Folic acid analogs

folate FH2 FH4

NADPH + H+

NADP+NADPH + H+

NADP+

FH2 reductase FH2 reductase

AP or MTX

- -

• the structural analogs of folic acid (e.g. MTX) are widely used to control cancer (e.g. leukaemia)

Notice: These inhibitors also affect the proliferation of normally growing cells. This causes many side-effects including anemia, baldness, scaly skin etc.

Degradation of purine nucleotides

N

HC

N

C

C

C

N

CH

N

NH2

Ribose-P

AMP

HN

HC

N

C

C

C

N

CH

N

O

Ribose-P

IMPHN

HC

N

C

C

C

NH

CH

N

O

HN

C

NH

C

C

C

NH

CH

N

O

O

HN

C

NH

C

C

C

NH

C

N

O

O

O

GMP

Hypoxanthine

Uric Acid Xanthine

Xanthine Oxidase

(2,6,8-trioxypurine)

Adenosine deaminase

Xanthine oxidase

The end product of purine metabolism

• 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 normal concentration of uric acid in the serum of adults is in the range of 3-7 mg/dl

Uric acid

• 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

Gout

• 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 uric acid and the gout

Uric acid

Over 8mg/dl, in the plasma

Gout, Urate crystallization

in joints, soft tissue, cartilage and kidney

Hypoxanthine

Xanthine Out of body

In urine

Diabetese nephrosis

……

Advanced Gout Clinically Apparent Tophi

1

1. Photos courtesy of Brian Mandell, MD, PhD, Cleveland Clinic.

2. Photo courtesy of N. Lawrence Edwards, MD, University of Florida.

3. ACR Clinical Slide Collection on the Rheumatic Diseases, 1998.

2 1

3

HN

HC

N

C

C

C

NH

CH

N

O

Hypoxanthine

HN

HC

N

C

C

C

NH

N

HC

O

Allopurinol

Allopurinol – a suicide inhibitor used to treat Gout

Xanthine oxidase

Xanthine oxidase

Synthesis of pyrimidine nucleotides

• shorter pathway than for purines

• pyrimidine ring is made first, then attached to ribose-P (unlike purine biosynthesis)

• only 2 precursors (aspartate and glutamine, plus HCO3

-) contribute to the 6-membered ring

• requires 6 steps (instead of 11 for purine)

• the product is UMP (uridine monophosphate)

De novo synthesis

N

C

N

C

C

C

12

34

5

6

Asp

CO2

Gln

Element source of pyrimidine base

• Carbamoyl phosphate synthetase (CPS) exists in 2 types:

• CPS-I, a mitochondrial enzyme, is dedicated to the

urea cycle and arginine biosynthesis

• CPS-II, a cytosolic enzyme, used here

It is the committed step in animals

Step 1: synthesis of carbamoyl phosphate

Step 2: synthesis of carbamoyl aspartate

ATCase: aspartate transcarbamoylase

Carbamoyl phosphate is an “activated” compound, so no energy input is needed at this step

Step 3: ring closure to form

dihydroorotate

Step 4: oxidation of dihydroorotate

to orotate

QH2

CoQ

(a pyrimidine)

Step 5: acquisition of ribose phosphate

moiety

Step 6: decarboxylation

of OMP

UDP

ADP

UTP

ATP ADP

UMP

ATP

kinase kinase

UTP and CTP biosynthesis

• the immediate precursor of thymidylate (dTMP) is dUMP

• the formation of dUMP either by deamination of dCMP or by

hydrolyzation of dUDP

dTMP dTDP dTTP

dUMP

dUDP dCMP dCDP

N5,N10-methylene-

tetrahydrofolic Acid

ATP ATP

ADP ADP

dTMP synthetase

UDP

Formation of dTMP

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, N

10-CH2-FH4 FH2

dTMP synthesis at the nucleoside monophosphate level

carbamoyl phosphate

carbamoyl aspartate

UMP

ATP + CO2 + Gln

PRPP

UTP CTP

ATP + R-5-P

purine nucleotide

pyrimidine nucleotide

Regulation of de novo synthesis

+ 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

Salvage pathway

• antimetabolites of pyrimidine nucleotides are similar with them of purine nucleotides

Antimetabolites of pyrimidine nucleotides

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

HN

NH

O

O

FHN

NH

O

O

CH3

thymine5-FU

Pyrimidine analogs

5-FU 5-FdUMP

5-FUTP

dUMP dTMP

Synthesis of RNA

Destroy structure of RNA

• Azaserine (AS) inhibits the synthesis of CTP

Amino acid analogs

• Methotrexate (MTX) inhibits the synthesis of dTMP

Folic acid 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

Nucleoside analogs

Degradation of pyrimidine nucleotides

H2OH2O

H2N CH2 CH2 COOH H2N CH2 CH COOH

CH3

N

NH

O

NH2

H2O NH3HN

NH

O

O

CH2

CH2NH2

NH

O

HOOC

HN

NH

O

O

CH3

CH2

CHNH2

NH

O

HOOC

CH3

cytosine uracilthymine

¦Â-ureidopropionate

¦Â-ureido-isobutyrate

CO2 + NH3

¦Â-alanine ¦Â-aminoisobutyrate

Highly soluble products

dATP

dGTP

AMP

GMP

ADP

GDP

dADP

dGDP

IMP

ATP

GTP

Summary of purine biosynthesis

CTP

UDP UTP

CDP

dUDP

dCDP

dUMP

dCMP

dTMP

UMP

dTDP dTTP

dCTP

Summary of pyrimidine biosynthesis

• Purines built up on ribose

– PRPP synthetase: key step

– First, synthesis IMP

• Pyrimidine rings built, then ribose added

– CPS-II: key step

– First, synthesis UMP

• Salvage is important

Summary of nucleotide biosynthesis