Medical Biochemistry

Mr. G. Siluchali

Levy Mwanawasa Medical

University
Metabolism of Nucleotides

N
N N

N N N
H
 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
 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
 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
 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.
 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-Formyltetrahydrofolate

N10-Formyltetrahydrofolate

First, synthesis Inosine-5'-Monophosphate, IMP

 FH4 (or THF)

N10—CHO—FH4
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
 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

• Once formed, IMP is rapidly

converted to AMP and GMP (it does
not accumulate in cells).
 N10-CHOFH4

N10-CHOFH4
 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

kinase kinase
AMP ADP ATP

ATP ADP ATP ADP

kinase kinase
GMP GDP GTP

ATP ADP ATP ADP

5. Regulation of de novo synthesis

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]
• 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
Lesch-Nyhan syndrome
 § 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

nucleotide 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

 § 2. 4 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.
1. Purine analogs
• 6-Mercaptopurine (6-MP) is an 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
2. Amino acid analogs
• Azaserine (AS) is a analog of Gln.

O NH2

H2N C CH2 CH2 CH COOH Gln

O NH2

N N CH2 C O CH2 CH COOH AS

 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
R＝ H： AP R＝ CH3： MTX
TXT

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

 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.
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
 Advanced Gout
Clinically Apparent Tophi
1 2

1 3

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.
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
 § 4.1 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
3. UTP and CTP biosynthesis

kinase kinase
UMP UDP UTP

ATP ADP ATP ADP

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.

UDP dUDP dCMP dCDP

dUMP
N5,N10-methylene-
tetrahydrofolic Acid
dTMP synthetase

ATP ATP
dTMP dTDP dTTP
ADP ADP
 dTMP synthesis at the nucleoside
monophosphate level.
dUDP
H2O
O O
Pi CH3
HN thymidylate synthase HN

O
NH3 O N N
5 10 FH2
d R 5' P N , N -CH2-FH4 d R 5' P
H2O
dUMP FH2 dTMP
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 those 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
¦Â-ureidopropionate
CH2 CH2 ¦Â-ureido-
O N O N
H H isobutyrate
H2O H2O

H2N CH2 CH2 COOH H2N CH2 CH COOH

CO2 + NH3
CH3
¦Â-alanine ¦Â-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