Nucleotides

Metabolism

Dr ASIFA MAJEED

ASSISTANT PROFESSOR
DEPARTMENT OF BIOCHEMISTRY AND
MOLECULAR BIOLOGY
ARMY MEDICAL COLLEGE RAWALPINDI
 HISTORY
Albrecht Kossel (1853-1927), German physiologist and Nobel
laureate

In1879, Kossel focused his studies on nuclein, a substance found

within the nucleus of a cell. Kossel determined that it was
composed partly of protein and partly of a nonprotein substance.
This second substance consisted of nucleic acids.
Nucleic acids consist of nitrogen-bearing compounds known as
purines and pyrimidines. From these purines and pyrimidines,
Kossel and his colleagues isolated the nitrogen-containing bases
cytosine, thymine, adenine, and guanine
 Nucleotide Metabolism

Biological Significance of Nucleotide

Metabolism

• Nucleotides make up nucleic acids (DNA and RNA)

• Nucleotide triphosphates are the “energy carriers” in
cells (primarily ATP)
• Many metabolic pathways are regulated by the level of
the individual nucleotides
– Example: cAMP regulation of glucose release
• Adenine nucleotides are components of many of the
coenzymes
– Examples: NAD+, NADP+, FAD, FMN, coenzyme A
 Nucleotide Metabolism

Medical significance of nucleotide

metabolism

• Anticancer agents:
• Rapidly dividing cells biosynthesize lots of
purines and pyrimidines, but other cells reuse
them. Cancer cells are rapidly dividing, so
inhibitor of nucleotide metabolism kill them
• Anti viral agents
 Nucleotide Metabolism

Nomenclature

Nucleotides are composed of:

 Nitrogenous base
 Pentose sugar
 Phosphate groups
 Nucleotide Metabolism

Nitrogenous Bases
• Aromatic and heterocyclic
• Derived from purine or pyrimidine
• Numbering of bases is “unprimed”
 Nucleotide Metabolism

Important Purines
Adenine and guanine are the principal purines of
both DNA and RNA.

Adenine Guanine
 Nucleotide Metabolism

Important Pyrimidines
Pyrimidines that occur in DNA are cytosine and
thymine. Cytosine and uracil are the pyrimidines in
RNA.

Thymine
Uracil Cytosine
 Nucleotide Metabolism

Sugars

• Pentoses (5-C sugars)

• Numbering of sugars is “primed”

Deoxyribose
Ribose
 Nucleotide Metabolism

Nucleosides

• Result from linking one of the sugars with a purine

or pyrimidine base through an N-glycosidic linkage

– Purines bond to the C1’ carbon of the sugar at

their N9 atoms
– Pyrimidines bond to the C1’ carbon of the sugar
at their N1 atoms
 Nucleotide Metabolism

Nucleosides
 Nucleotide Metabolism

Nucleotides
• Result from linking one or more phosphates with a
nucleoside onto the 5’ end of the molecule
through esterification
 PYRIMIDINE
BIOSYNTHESIS

Pyrimidine is
synthesized
from
carbamoyl phosphate
and
aspartate
P 1: Carbamoyl phosphate synthesis in the cy

carbamoyl phosphate synthetase II

EP 2: Aspartate transcarbamoylase cataly
formation of carbamoylaspartate

committed step
STEP 3: Ring closure
STEP 4: PRPP addition
 STEP 5: UTP formation

UMP kinase
UMP + ATP UDP + ADP

Nucleoside diphosphate kinase

UDP + ATP UTP + ADP

XDP + YTP XTP + YDP

Nucleoside mono-, di and triphosphates are interconvertable

TEP 6: Amination of UTP results in CTP

CTP synthetase
 Hereditary Orotic
Aciduria
• an inherited human disease caused by a
deficiency in the multifunctional enzyme
that catalyzes the last 2 steps in the
pyrimidine synthesis
• Defect in de novo synthesis of pyrimidines
• Loss of functional UMP synthetase
– Gene located on chromosome III
• Characterized by excretion of orotic acid
• Results in severe anemia and growth
retardation
• Extremely rare (15 cases worldwide)
• Treated by feeding UMP
 PURINE BIOSYNTHESIS
First purine derivative formed is Inosine Mono-phosphate
(IMP)
• The purine base is hypoxanthine
• AMP and GMP are formed from IMP
 PURINE BIOSYNTHESIS
De Novo
Purine is
synthesized
from
amino acids,
tetrahydrofolate
and CO2
The committed step
in de novo purine
synthesis is the
activation of PRPP
to phospho-
ribosylamine
 C-STEP: PRPP activation

PRPP + Phosphoribosyl-
Glutamine amine +
Glutamate
STEP 1: Addition
of glycine
STEP 2: Formylation
by N10 -formyltetra-
hydrofolate

formyltransferase
STEP 3: Transfer of
nitrogen from
glutamine
before ring closure)

4
STEP 4: Dehydration
and ring closure

-H2O
 STEPS 5-8:
Carboxylation
Aspartate addition
Formylation
Dehydration and
ring closure
STEP 9: Conversion
of IMP to ATP
and GTP
STEP 9: Conversion
of IMP to ATP
and GTP
 Salvage Pathway for
Purines

Hypoxanthine
or + PRPP = IMP or GMP + PPi
Hypoxanthineguanosylphosphoribosyl transferase
Guanine (HGPRTase)

Adenine + PRPP = AMP + PPi

Adeninephosphoribosyl transferase
(APRTase)
 Specific Kinases Convert
NMP to NDP
Nucleoside Nucleoside
Monophosphates Diphosphates
Monophosphate
Kinases
• Monophosphate kinases are specific for the bases

Adenylate Kinase
AMP + ATP 2ADP

Guanylate Kinase
GMP + ATP GDP + ADP
 Lesch-Nyhan syndrome
• there is a defect or lack in the HGPRT
enzyme
• the rate of purine synthesis is increased
about 200X
• X-linked syndrome
• uric acid level rises and there is gout
• in addition there are mental aberrations
• patients will self-mutilate by biting lips and
fingers off
Regulation of purine biosynthesis
 CATABOLISM OF
PURINES
adenase
ADENINE + H 2O HYPOXANTHINE + AMMONIA

guanase XANTHINE + AMMONIA

GUANINE + H 2O

xanthine
HYPOXANTHINE + O 2 + H 2O oxidase XANTHINE + H 2O2
xanthine
XANTHINE + O 2 + H 20 URIC ACID + H 2O2
oxidase
 GOUT
• a disorder associated with abnormal amounts
of urates in the body
• early stage: recurring acute arthritis
• late stage: chronic deforming arthritis and
eventual renal complication
• disease with rich history dating back to
ancient Greece
• prevails mainly in adult males
• symptoms are cause by deposition of crystals
of monosodium urate monohydrate
Gout
 Therapy of acute gout
• treat with colchicine
• avoid aspirin
• uric acid lowering agents should
never be started or stopped during
acute attack
• pain resolution occurs within 48-72
hrs
 Immunodeficiency Diseases
Associated with Purine
Degradation
• Defect in adenosine deaminase
– Removes amine from adenosine
• SCID- severe combined
immunodeficiency
• Defect in both B-cells and T-cells
(Disease of Lymphocytes)
• Patients extremely susceptible to
infection
 Thymidylate (dTMP) can be synthesized from either CDP or UDP
NH2
ATP ADP
C
N CH CDP dCDP dCTP
H2O
C CH nucleoside
O N ribonucleotide dCTP
diphosphate
reductase deaminase
kinase
NH3
O UDP dUDP dUTP
C
HN CH ATP ADP H2O

C CH
O- O N PPi
…-O-P-O-CH
2
O dUMP
O
N5,N10 -methylene-
OH OH O THF
C CH3 thymidylate
HN C synthase
dTMP
C CH
O N
ATP dTTP
 Degradation of Pyrimidines
• CMP and UMP degraded to bases
similarly to purines
– Dephosphorylation
– Deamination
– Glycosidic bond cleavage
• Uracil reduced in liver, forming β -
alanine
– Converted to malonyl-CoA  fatty acid
synthesis for energy metabolism
 Regulation of
Pyrimidine Biosynthesis
• Regulation occurs at first step in the pathway ()

• 2ATP + CO2 + Glutamine = carbamoyl phosphate

X
Inhibited by UTP
 H
H2N N N
Enzymes of nucleotide methotrexate
biosynthesis provide targets N
N O
for cancer chemotherapy NH2 CH3 N C-NH-Glu

O
C Analog of DHF. Inhibits
O HN C-F dihydrofolate reductase
C
C-F (in vivo) C CH
HN O- O N
C CH
-
O-P-O-CH2
O N O
O Analog of dUMP. Inhibits
H
thymidylate synthase.
5-fluorouracil FdUMP
OH
CO2-
CO2 -

Analog of Gln. Inhibits glutamine

+
H3N C H
azaserine +
H3N C H
(O-diazoacetyl- CH2 amidotransferases (steps 1 & 4 of CH2
L-serine) purine biosynthesis, CTP synthase, & CH2
O
- + carbamoylphosphate synthetase II). H2N-C=O
N=N=CH-C-O-C=O
O Gln
 FORMATION OF
DEOXYRIBONUCLEOTIDES
• Ribonucleotide reductase studied by
Joanne Subbe
• very complex enzyme; contains:
• Tyrosine radical
• Two catalytically active cysteine residues
• Cys are reduced by other proteins –
thioredoxin
• Ribo. Reductase is the therapeutic target of
the anticancer drug hydroxyurea
 Ribonucleotide Reductase
• The enzyme system consists of 4 proteins
– Two of which constitute the Ribonucleotide
Reductase (α 2β 2)
– Thioredoxin and thioredoxin reductase
• Has three different nucleotide-binding sites
– Substrate: NDPs
– Activity-determining: ATP & dATP
– Specificity-determining: ATP, dTTP, dGTP,
and dATP
 Conversion of
Ribonucleotides to
Deoxyribonucleotides
1) A free radical group on the ribonucleotide reductase removes a
hydrogen atom from carbon 3' of ribose and forms a free radical
on the ribose of the nucleotide.

2) A thiol group of the enzyme donates a proton to the hydroxy

group on carbon 2 followed by the elimination of a molecule of
water.

4) Carbon 2 is reduced by the second sufhydryl group.

5) The enzyme donates a hydrogen atom to the free radical

(generated in step 1) on carbon 3 to form the
deoxyribonucleotide.

The enzyme is converted to the original free radical form and must
be reduced by thioredoxin to its starting disulfhydryl form.
 1

5 deoxyribonucleoside
Conversion of Ribonucleotides
to Deoxyribonucleotides

HOCH 2 O O H
BASE HOCH2 O OH
BASE
5´ 5´
4´ H H 1´ 4´ H H 1´
H 3´ 2´ H H H
3´ 2´
Ribonucleotide HO OH
HO H Reductase
Deoxyribonucleoside Ribonucleoside
 Regulation of ribonucleotide reductase
The regulation occurs by binding of ribo-NTPs to either
the general activity sites or to the specificity sites of
the enzyme.
The binding of ATP at activity sites leads to increased
enzyme activity, while low affinity binding of dATP
inhibits the enzyme. To a minor degree dGTP and other
dNTP also inhibit ribonuclease.

The specificity sites bind ATP, dATP, dGTP, or dTTP

with high affinity. The binding of nucleotides at
specificity sites effectively allows the enzyme to detect
the relative abundance of the four dNTPs and to adjust
its affinity for the synthesis of the less abundant
dNTPs, to synthesize a balance proportion of dNTP.
 NMP to NDP/NTP
• Monophosphate kinases are specific for
the bases Adenylate Kinase
AMP + ATP 2ADP

Guanylate Kinase
GMP + ATP GDP + ADP

UMP kinase
UMP + ATP UDP + ADP

Nucleoside diphosphate kinase

UDP + ATP UTP + ADP
 Specific Drug
Resistance
Methotrexate
• Methotrexate works by inhibiting the function of dihydrolfolate
reductase (DHFR)
• Cells develop ways to avoid this block
– Mutations in DHFR that make it bind less tightly to MTX
– Amplication of the DHFR gene (more enzyme activity)
 AZT as an Anti-HIV
Agent
• Azido-3’-deoxythymidine
• Pyrimidine Analogue O
C CH3
• HIV is a retrovirus HN C
• RNA genome that is reverse- O C
N
C
H
HOCH2 O
transcribed to DNA H H
• Viral polymerase is inhibited H H
by AZT N3 H
READING REFERENCES

Lippincott Biochemistry
Harper Biochemistry
Stryer Biochemistry
THANK
YOU