Metabolism of Nucleotides

N
N N

N N N
H
 Nucleoside and Nucleotide

Nucleoside = Nitrogenous base ribose

Nucleotide = Nitrogenous base ribose phosphate

Purines vs Pyrimidines
Structure of nucleotides
Pyrimidine OR Purine

N--glycosyl
bond

Ribose
or
2-deoxyribose
Degradation of nucleic acid
Nucleoprotein
In stomach Gastric acid and pepsin

Nucleic acid Protein

In small intestine Endonucleases: RNase and DNase

Nucleotide
Nucleotidase

Phosphate Nucleoside
Nucleosidase

Base Ribose
 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
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.
 De novo synthesis
Site:
in cytosol of liver, small intestine and thymus
Characteristics:
a. Purines are synthesized using 5-
phosphoribose(R-5-P) as the starting material
step by step.
b. PRPP(5-phosphoribosyl-1-pyrophosphate) is
active donor of R-5-P.
c. AMP and GMP are synthesized further at the
base of IMP(Inosine-5'-Monophosphate).
 Element sources of purine bases

N10Formyltetrahydrofolate

N10Formyltetrahydrofolate

First, synthesis Inosine-5'-Monophosphate, IMP

 Synthesis of Inosine Monophosphate
(IMP)

Basic pathway for biosynthesis of purine

ribonucleotides
Starts from ribose-5-phosphate(R-5-P)
Requires 11 steps overall
occurs primarily in the liver
 OH Step 1: Activation of ribose-5-phosphate

Committed step
ATP
1 ribose phosphate pyrophosphokinase
AMP

Step 2: Acquisition of purine atom N9

2
Gln:PRPP amidotransferase

Steps 1 and 2 are tightly

regulated by feedback inhibition
Step 3: Acquisition of purine atoms C4, C5, and N7

3
glycinamide synthetase
Step 4: Acquisition of purine atom C8

4
GAR transformylase
Step 5: Acquisition of purine atom N3

5
Step 6: Closing of the imidazole ring

6
Step 7: Acquisition of C6

7
AIR carboxylase

Carboxyaminoimidazole
ribonucleotide (CAIR)
Step 8: Acquisition of N1

Carboxyaminoimidazole
ribonucleotide (CAIR)

SAICAR synthetase
Step 9: Elimination of fumarate

adenylosuccinate lyase
Step 10: Acquisition of C2

AICAR transformylase
Step 11: Ring closure to form IMP

IMP Synthase

Once formed, IMP is rapidly

converted to AMP and GMP (it does
not accumulate in cells).
 Conversion of IMP to AMP and GMP

Adenylosuccinate
lyase
Adenylosuccinate
synthetase

Note: ATP is used for

GMP synthesis.

IMP
dehydrogenase
XMP-GLUTAMATE
amidotransferase

IMP is the precursor for both AMP and GMP.

4. ADP, ATP, GDP and GTP biosynthesis

kinase kinase
AMP ADP ATP

ATP ADP ATP ADP

kinase kinase
GMP GDP GTP

ATP ADP ATP ADP

Purine nucleotide biosynthesis is regulated by feedback
inhibition
 Salvage pathway
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.
 Purine Salvage Pathway
.
adenine
phosphoribosyl transferase
Adenine AMP

PRPP PPi
O

O N N
N N 2-O N
3POH2C O
N
N hypoxanthine-guanine
N phosphoribosyl transferase
Hypoxanthine (HGPRT) HO OH
IMP
O O
PRPP PPi
N N N N
N N NH2 2-O N
3POH2C O
N NH2
Guanine

HO OH
GMP
.
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

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.
P P O CH2 O Base P P O CH2 O Base
ribonucleotide
reductase
Mg2+
OH OH H 2O OH H
thioredoxin SH thioredoxin S
NDP dNDP
SH S ATP
N=A, G, C, U
+ FAD + kinase
NADP NADPH + H
thioredoxin ADP
reductase
dNTP

Deoxyribonucleotide synthesis at the NDP level

 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 DNA, RNA, and proteins.
Widely used to control cancer.
Purine analogs
6-Mercaptopurine (6-MP) is a analog of
hypoxanthine.
 6-MP nucleotide is a analog of IMP
de novo synthesis
-
amidotransferase
-
IMP
6-MP 6-MP nucleotide
-

- AMP and GMP

HGPRT
-
salvage pathway
Amino acid analogs
Azaserine (AS) is a analog of Gln.
Folic acid analogs
Aminopterin (AP) and Methotrexate (MTX)

MTX
 NADPH + H+ NADPH + H+
NADP+ NADP+

folate FH2 FH4

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.
Catabolism of
purine
nucleotide
 Uric acid
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.
 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 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.
Xanthine oxidase inhibitors inhibit
production of uric acid, and treat gout
The uric acid and the gout Hypoxanthine

Out of body Xanthine

In urine
Uric acid
Over 8mg/dl, in the plasma
Diabetese nephrosis
Gout, Urate crystallization
in joints, soft tissue, cartilage and kidney
Allopurinol a suicide inhibitor used to treat Gout
O O
C C H
N C
HN C HN C
CH N
HC C HC C
N N N
N H H
Hypoxanthine Allopurinol

Xanthine oxidase

Xanthine oxidase
 Pyrimidine Biosynthesis
De novo synthesis
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)
1. Element source of pyrimidine
base

C
Gln 4
N3 5C
Asp
CO2 C2 6C
1
N
 Step 1: Synthesis of carbamoyl
phosphate

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 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

CoQ

QH2

(a
pyrimidine)
Step 5: Acquisition of ribose phosphate moiety

Step 6: Decarboxylation of OMP

The big picture
UTP and CTP biosynthesis

kinase kinase
UMP UDP UTP

ATP ADP ATP ADP

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.
dUDP
H2O
O O
Pi CH3
HN thymidylate synthase HN

O
NH3 O N N

d R 5' P d R 5' P
H2O
dUMP FH2 dTMP
dCMP NADPH
reductase
+ H+

NADP+
 Salvage pathway

uridine uridine-cytidine kinase UMP + ADP

cytidine + ATP CMP
thymidine kinase
deoxythymidine + ATP dTMP + ADP

deoxycytidine kinase
deoxycytidine + ATP dCMP + ADP

pyrimidine phosphate
uracil ribosyltransferase UMP
thymine + PRPP dTMP + PPi
orotic acid OMP
 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.

O O
F CH3
HN HN

O N O N
H H

5-FU thymine
2. Amino acid analogs
Azaserine (AS) inhibits the synthesis of
CTP.

3. Folic acid analogs

Methotrexate (MTX) inhibits the
synthesis of dTMP.
4. Nucleoside analogs
Arabinosyl cytosine (ara-c) inhibits
the synthesis of dCDP.
NH2 NH2

N N

O N O N
CH2OH CH2OH
O O

H OH H H
H H H H
OH H OH OH
ara-c cytosine
 NH2 O O
H2O NH3 CH3
N HN HN
O N O N O
H H N thymine
uracil H
cytosine

HOOC HOOC
NH2 CH2 NH2 CH CH3
-ureidopropionate
CH2 CH2 -ureido-
O N O N
H H isobutyrate
H2O H2O

H2N CH2 CH2 COOH H2N CH2 CH COOH

CO2 + NH3
CH3
-alanine -aminoisobutyrate
Highly soluble products
 Summary of purine biosynthesis

dADP dATP

AMP ADP ATP

IMP
GMP GDP GTP

dGDP dGTP
Summary of pyrimidine biosynthesis
dTMP dTDP dTTP

dUMP dUDP

UMP UDP UTP

CDP CTP

dCMP dCDP dCTP

Summary of Nucleotide Synthesis

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
 Points
Synthesis of Purine Nucleotides
De novo synthesis: Site, Characteristics, Element sources of
purine bases
Salvage pathway: definition, significance, enzyme, Lesch-
Nyhan syndrome
Formation of deoxyribonucleotide
Antimetabolites of purine nucleotides:
Purine, Amino acid, and Folic acid analogs
Degradation of Purine Nucleotides
Uric acid, gout
Synthesis of Pyrimidine Nucleotides
De novo synthesis: Characteristics, Element sources of
pyrimidine bases
Salvage pathway
Antimetabolites of pyrimidine nucleotides
Catabolism of Pyrimidine Nucleotides