Biochemistry

Metabolism of Nucleotides

Department of BMB
 Biological functions of nucleotides

1. Building blocks of nucleic acids (DNA and RNA).

2. Involved in energy storage, muscle contraction,
active transport, maintenance of ion gradients.
3. Activated intermediates in biosynthesis
(e.g. UDP-glucose, S-adenosylmethionine).
4. Components of coenzymes (NAD+, NADP+, FAD,
FMN, and CoA)
5. Metabolic regulators:
a. Second messengers (cAMP, cGMP)
b. Phosphate donors in signal transduction (ATP)
c. Regulation of some enzymes via adenylation and
uridylylation
 Contents
Review: Structure of nucleic acid
Degradation of nucleic acid
Synthesis of Purine Nucleotides
Degradation of Purine Nucleotides
Synthesis of Pyrimidine Nucleotides
Degradation of Pyrimidine Nucleotides
Metabolism of Nucleotides

Nitrogenous-base
 Nucleoside and Nucleotide
Nitrogenous base ribose
Nucleoside =
Nitrogenous base ribose phosphate
Nucleotide =
Purines vs Pyrimidines
Structure of nucleotides
pyrimidine OR purine

N-b-
glycosyl
bond

Ribose
or
2-deoxyribose
 Section 1
Degradation of nucleic acid
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
 Section 2

Synthesis 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, and one-carbon
units.

Salvage pathways: The synthesis of nucleotide

by recycle the free bases or nucleosides released
from nucleic acid breakdown.
 2.1 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).
1. Element sources of purine bases

N10-
Formylt
etrahydr
N10- ofolate
Formylte
trahydro
folate

First, synthesis Inosine-5'-Monophosphate, IMP

 FH4 (or THF)

N10CHOFH4
2. 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
1O
AA
H
TM
PP Step 1:Activation of ribose-5-phosphate

Committed step
2
ribose phosphate pyrophosphokinase

Step 2: acquisition of purine atom N9

Gln:PRPP amidotransferase

Steps 1 and 2 are tightly

regulated by feedback inhibition

5-PRA
 3
Step 3: acquisition of purine atoms C4, C5, and N7

glycinamide synthetase
 4

Step 4: acquisition of purine atom C8

GAR transformylase
 5
Step 5: acquisition of purine atom N3
Step 6: closing of the imidazole ring
 7
a
r
Step
b 7: acquisition of C6
o
x
y
a
m
AIR carboxylase
i
n
o
i
m
i
d
a
z
o
l
e
 oxya
Step 8: acquisition
minoi of N1
mida
zole
ribon
ucleo
tide
(CAI
R)

SAICAR synthetase
Step 9: elimination of fumarate

adenylosuccinate lyase
Step 10: acquisition of C2

AICAR transformylase
Step 11: ring closure to form IMP

Once formed, IMP is rapidly

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

Note: GTP is used for AMP synthesis.

Note: ATP is used for

GMP synthesis.

IMP is the precursor for both AMP and GMP.

4. ADP, ATP, GDP and GTP biosynthesis

k k
i i
AMPn ADP n ATP
a a
ADP ATP ADP
sATP s
e e
k k
i i
GMPn GDP n GTP
a a
sATP ADP ATP
s ADP
e e
 Purine nucleotide biosynthesis is regulated by feedback
inhibition
 2.2 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
 2. 3 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 H2O 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

1. Purine analogs
6-Mercaptopurine (6-MP) is a analog of
hypoxanthine.

OH SH
N N
N N

N N N N
H H
hypoxanthine 6-MP
 6-MP nucleotide is a analog of IMP
de novo synthesis
-
amidotransferase
-
IMP
6-MP 6-MP nucleotide -

AMP and GMP

-
HGPRT

-
salvage pathway
 3. Folic acid analogs
Aminopterin (AP) and Methotrexate (MTX)

NH2 R O COOH
N
N CH2 N C NH C CH2 CH2 COOH
H
H2N N N
RHAP RCH3TXT
MTX

OH H O COOH
N
N CH2 N C NH C CH2 CH2 COOH
H
H2N N N
folic acid
 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.
 Section 3
Degradation of Purine Nucleotides
 NH2 O
Adenosine
C C
N Deaminase N
N C HN C
CH CH
HC C HC C O
N N N
N
C N
Ribose-P Ribose-P
HN C
IMP CH
AMP
HC C
N N
Xanthine Oxidase H
Hypoxanthine
O O
C N C N
HN C HN C
C O CH
C C C C
O N N O N N
H H H H
GMP
Uric Acid Xanthine

(2,6,8-trioxypurine) The end product of purine metabolism

 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.
 H
Xanthine
y
The uric acid and the gout p
o
x
Out of body a
n
In urine t
Uric acidh
Over 8mg/dl, ini the plasma
Diabetese nephrosis n
e
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
 Section 4
Synthesis of Pyrimidine Nucleotides
1. Element source of pyrimidine
base

C
4
Gln N3 5C
Asp
CO 2 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 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
3. UTP and CTP biosynthesis
UMP A A kUTP
Akin
UDP A
TaseD T i D
P P P nP
a
s
e
 4. Formation of dTMP
The immediate precursor of thymidylate (dTMP) is dUMP.
The
UN5,d dformation
d d of dUMP either by deamination of dCMP or
dT
DN10Ubyd hydrolyzation
AC
U MP d
AdAAC of dUDP. The former is the main route.
P - D MsynTMDTTDD
P T
met P
PP PPT
P
P thetD
hyle ase P T
ne-
M
tetr P P
ahy
drof
olic
Aci
d
dUDP
H2O

Pi
HN
dTMP synthesis at the nucleoside
O

thymidylate synthase HN
O
CH3

O
NH3 O N

monophosphate level.
N
5 10 FH2
d R 5' P N , N -CH2-FH4 d R 5' P
H2O
dUM P FH2 dT M P
dCMP NADPH
reductase
+ H+
FH4
NADP+
 4. 2 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
 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.

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
 Section 5
Degradation of Pyrimidine Nucleotides
 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: NDP level
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