BIOCHEMISTRY

Nucleotide Metabolism
Dr.Thomas B.Nyambo
Dept of Biochemistry
School of Medicine
MUCHS
23rd November 2002
 STRUCURE OF NUCLEOTIDES
Nucleotides consist of three parts:
1. A five-carbon sugar (hence a pentose). Two kinds are found:
Deoxyribose, which has a hydrogen atom attached to its #2
carbon atom (designated 2')
Ribose, which has a hydroxyl group atom there
Deoxyribose-containing nucleotides, the deoxyribonucleotides,
are the monomers of DNA and ribose-containing nucleotides,
the ribonucleotides, are the monomers of RNA.
2.A nitrogen-containing ring structure called a base. The base is
attached to the 1' carbon atom of the pentose. In DNA, four
different bases are found:
i. two purines, called adenine (A) and guanine (G)
ii. two pyrimidines, called thymine (T) and cytosine (C)
1. Phosphate
 Purines and pyrimidines
.
 Composition of Nucleic acids
. Base Nucleoside Nucleotides

Adenine (A) Deoxyadenosine dAMP dADP dATP

Guanine (G) Deoxyguanosine dGMP dGDP dGTP

DNA
Cytosine (C) Deoxycytidine dCMP dCDP dCTP

Thymine (T) Deoxythymidine dTMP dTDP dTTP

Adenine (A) Adenosine AMP ADP ATP

Guanine (G) Guanosine GMP GDP GTP

RNA
Cytosine (C) Cytidine CMP CDP CTP

Uracil (U) Uridine UMP UDP UTP

Hydrolysis of Polynucleotides
.
 Dietary nucleoproteins
The nucleotides are hydrolyzed by
nucleotidases to give the nucleosides and
Pi. This is probably the end product in the
intestine with the nucleosides being the
primary form absorbed.
In some tissues, phosphorylases to yield
the base and ribose 1-P (or deoxyribose 1-
P The sugar phosphate can either be
reincorporated into nucleotides or
metabolized via the PPP.
The purine and pyrimidine bases released are either
degraded or salvaged for reincorporation into
nucleotides.
 Dietary nucleotides

.
 Dietary nucleoproteins
Dietary nucleoprotein is degraded by
pancreatic enzymes and tissue nucleoprotein
by lysosomal enzymes. After dissociation of
the protein and nucleic acid, the protein is
metabolized like any other protein.
The nucleic acids are hydrolyzed by
nucleases to yield a mixture of
polynucleotides. These are further cleaved
by phosphodiesterases (exonucleases) to a
mixture of the mononucleotides.
The pancreatic nucleotidases gives the 3'-nucleotides and
that of the lysosomal nucleotidases gives 5'-nucleotides.
 Nucleotide Metabolism
Many organisms can synthesize purine and
pyrimidine nucleotides from low-molecular-
weight precursors
These de novo pathways are essentially
identical in all organisms.
Nucleotides can also be synthesized from the
partial breakdown of previously synthesized
nucleotides.
These pathways are called salvage pathways.
 Synthesis of PRPP(5-Phosphoribosyl-1-pyrophosphate
(PRPP).

.
 The first reaction
1.Inosinemonophosphatesynthesizeddenovoby
addingontoribosephosphate
2.Firststepandregulatedstepisconversionof
ribose5phosphatetophosphoribose1
pyrophosphate(PRPP).

 The first and second reactions

.
Thepyrophosphate'activates'theC1ontheriboseforfurtheraddition

 Subsequent reactions
.
 Further reactions
.
 De novo Synthesis of Purines

.
 Synthesis of GMP and AMP from Inosinic acid

.
 Synthesis of GMP and AMP from Inosinic acid

.
 Precursors to the purine ring

.
 Purine and pyrimidine
biosynthesis
.
 Interconvention of the nucleotides
.
Regulation of Purine Nucleotide Synthesis

The essential rate limiting steps in purine biosynthesis occur at the

first two steps of the pathway. The synthesis of PRPP by PRPP
synthetase is feed-back inhibited by purine-5'-nucleotides
(predominantly AMP and GMP).
Combinatorial effects of those two nucleotides are greatest, e.g.,
inhibition is maximal when the correct concentration of both adenine
and guanine nucleotides is achieved.
The amidotransferase reaction catalyzed by PRPP amidotransferase
is also feed-back inhibited allosterically by binding ATP, ADP and
AMP at one inhibitory site and GTP, GDP and GMP at another.
Conversely the activity of the enzyme is stimulated by PRPP.
Additionally, purine biosynthesis is regulated in the branch pathways
from IMP to AMP and GMP. The accumulation of excess ATP leads to
accelerated synthesis of GMP, and excess GTP leads to accelerated
synthesis of AMP
.
Salvage of purine and pyrimidine bases

.
 Degradationof of purine nucleotides to uric acid

.
 Degradationof of purine nucleotides to uric acid

.


 Catabolism of uric acid to ammonia and CO2

.
 Uric acid
Hyperuricemia is a condition characterized by
chronic elevation of blood uric acid levels.
This condition is known as gout.
High levels of urate leads to precipitation of
sodium urate in the synovial fluid of joints.
Precipitates can cause inflammation, arthritis,
and sometimes a severe degeneration of the
joints.
 Enzymatic causes of Gout
PRPP synthetase: Defects in PRPP synthetase may render
it insensitive to feedback inhibition by purine nucleotides.
PRPP amidotransferase: Defects in PRPP amidotransferase
may render it insensitive to feedback inhibition by purine
nucleotides leading to the overproduction of purine
nucleotides, excessive uric acid synthesis, and gout.
Hypoxanthine-Guanine Phosphoribosyltransferase
(HGPRT): HGPRT is a salvage pathway enzyme for purine
metabolism. The enzyme activity is not completely missing
in gout patients. Complete absence of the enzymatic activity
is associated with Lesch-Nyhan syndrome.
Defects in excreting uric acid due to the inability of the
kidney tubules to secrete uric acid.
Cancer patients on chemotherapy may experience gout
due to generation of many purines by nucleic acid
degradation after cell death.
 Treatment of gout

Allopurinol is a drug that is used to treat gout.

It is similar to hypoxanthine and inhibits xanthine oxidase, leading to
accumulation of hypoxanthine and xanthine, both of which are more
soluble and more readily excreted than uric acid.
 Lesch-Nyhan syndrome

Hypoxanthine-guanine phosphoribosyltransferase
(HGPRT) is a salvage pathway enzyme for purine
metabolism.
When a defect in HGPRT reduces its activity to a
low level, gout results.
Complete absence of activity of HGPRT, Lesch-
Nyhan syndrome results.
HGPRTgene is located on the X chromosome,
so the disease is sex-linked.
Affected individuals have severe gouty arthritis
and a malfunction of the nervous system with
neuropschiatry sumptoms
They rarely live beyond 20 years.
 ADA
.
 ADA

.
 Immunodeficiency in defective Purine Catabolism
Patients with severe combined immune deficiency
(SCID) are completely unable to mount an immune
response following an antigenic challenge.
Both the B and T lymphocytes are affected. The disease
arises from an inherited lack of adenosine deaminase
(ADA).
The absence of ADA allows dATP to accumulate from
the degradation of DNA.
High dATP levels inhibit production of the other dNTPs
needed for DNA replication because of their allosteric
effects on the enzyme ribonucleotide reductase.
Since white blood cells must proliferate for an immune
response to occur, and proliferations requires ample and
rapid synthesis of DNA proliferation is hampered.
 Pyrimidines

.
De novo synthesis of pyrimidine nucleotides

The first step is the formation of carbamoyl

aspartate from aspartate and carbamoyl
phosphate,
It is the primary regulatory point in the pathway.
Aspartate transcarbamoylase (ATCase) is
activated by ATP and inhibited by CTP which is
the end product of the pathway.
Another point of regulation is CTP synthetase,
which is feedback inhibited by CTP and
activated by GTP.
.
De novo synthesis of pyrimidine nucleotides

.
 De novo synthesis of pyrimidine nucleotides
.
 De novo synthesis of pyrimidine nucleotides

.
 Amidation of UTP
.
 Sources of atoms compared

.
 Reduction of ribonucleotides to deoxyribonucleotides
.
 Thymidylate synthesis
.
 Control of pyrimidine metabolism

.
 Cancer chmotherapy
Thymidylate synthesis is catalysed by thymidylate synthase
First UDP converted to dUDP by ribonucleotide reductase
Then dUDP dephosphorylated to dUMP
A methyl group is donated by THF to dUMP to make dTTP
Dihydrofolate (DHF) must be regenerated by a reaction
catalysed by Dihydrofolate reductase
Formation of dTTP is essential step for the biosynthesis of DNA
The anticancer drugs methotrexate inhibits dihydrofolate
reductase, which lowers the amount of dTMP that is
synthesized
Methotrexate resembles DHF and therefore targets cancer cells
that are rapidly proliferating and carrying out DNA synthesis
 De novo Synthesis of Pyrimidines
.
 Nucleotide Salvage
Salvage refers to the reuse of parts of nucleotides in
resynthesizing new nucleotides.
Enzymes in the salvage of nucleotides:
1. Phosphoribosyl transferases, which interconvert free bases plus
PRPP with nucleoside monophosphates;
2. Nucleotidases.It cleaves phosphates from nucleoside
monophosphates to form free nucleosides
3. Nucleoside kinases.Phosphorylate nucleosides to nucleoside
monophosphates
4. Phosphorylases.It uses phosphate to separate the base from
ribose, forming free bases and ribose-1-phosphate.
5. Phosphodiesterases, which convert oligonucleotides to
nucleoside monophosphates
6. Endonucleases. It convert nucleic acids to oligonucleotides
 Phosphoribosyl Transferases

Phosphoribosyl transferases are

enzymes involved in the salvage
pathways.
PRPP + base NMP + PPi,
i. where NMP is a nucleoside
monophosphate, such as AMP.
ii. e.g PRPP + Adenine AMP + PPi
 HGPRT
.
 Nucleotidases

Nucleotidases are enzymes that

hydrolyze a phosphate from a nucleoside
monophosphate to yield a nucleoside and
a free phosphate.
e.g CMP + H2O Cytidine + Pi
 Deoxyribonucleoside kinases
Purine salvage usually involves phosphoribosyltransferase
reactions. Another salvage route to making
deoxyribonucleotides is via deoxyribonucleoside kinases.
Human cells have four different deoxyribonucleoside kinases:
1. Thymidine kinase (located in the cytosol) - phosphorylates
deoxythymidine
2. Deoxycytidine kinase (located in cytosol) - phosphorylates
deoxycytidine
3 Deoxyguanosine kinase (located in mitochondria)
4. Thymidine kinase (located in mitochondria)
Mitochondrial thymidine kinase will also act on the anti-HIV
drug, 3'-azido-2'3'-dideoxythymidine (AZT). That is, the enzyme
can phosphorylate AZT to a deoxyribonucleotide of
azidothymidine, which is then incorporated into DNA.
 Catabolism of Pyrimidines
.
 Target Enzyme for Chemotherapy

.
 Cancer chemotherapy
.
 Nucleotide Analogs in chemotherapy

DNA polymerases and/or nucleotide

metabolism enzymes are the targets of
designer drugs.
Target enzymes include the following:
i. Deoxypyrimidine kinase of herpes virus .This
viral enzyme readily phosphorylates the drugs
5-iododeoxyuridine, acycloguanosine
(acyclovir, and ganciclovir. The viral DNA
polymerase attempts, in turn, to incorporate
them into DNA in place of the corresponding
dNTP. so DNA replication and virus growth are
inhibited selectively in infected cells.
 Nucleotide Analogs in chemotherapy
i. DNA polymerases.Arabinosyladenine (araA)
and arabinosylcytosine (araC) are readily
converted to triphosphates (araATP and
araCTP).
AraATP is a selective inhibitor of the DNA
polymerases of herpesvirus.
araC is used in chemotherapy and functions
by the same mechanism of inhibition on the
cellular polymerase.
 Nucleotide Analogs in chemotherapy
iii Reverse Transcriptase. 3'-Azido-2'3'-
dideoxythymidine (AZT), when converted
to the corresonding 5' triphosphate in
cells, is an inhibitor of the HIV reverse
transcriptase enzyme.
Other nucleoside analogs, such as 2'3'-
dideoxycytidine (ddC), 2'3'-
dideoxyinosine (ddI), and 2'3'-didehydro-
3'-deoxythymidine (d4T)
 Nucleotide Analogs in chemotherapy

.
 Nucleotide Analogs in chemotherapy

iv Dihydrofolate Reductase regenerates

tetrahydrofolate from the dihydrofolate
generated in the synthesis of dTMP from
dUMP.
Inhibitors of dihydrofolate reductase, such
as methotrexate and trimethoprim, inhibit
production of dTTP in susceptible species
 .

Interrelationship between thymidylate synthase and enzymes of tetrahydrofolate

metabolism.
 Sulfa Drugs

.
PYRIMIDINES
 CYTIDINE
.
 NUCLEOTIDES