11.

Synthesis and Degradation of

Nucleotides

Yohannis. W (M.Sc Biochem)

AAU, CHS,DB
 Introduction
 The genetic information is stored in DNA or RNA (RNA
viruses).
 The central dogma of biochemistry; that is the flow of
genetic information from DNA to DNA from DNA to RNA,
and then to protein.
Traditional dogma Extended dogma
 Role of Nucleotides
 Subunits of genetic material nucleic acids (DNA and RNA)
 Main energy currency in metabolic transactions (mostly ATP)
 Partially regulate metabolic pathways
 Mediate many hormomal signals
(cAMP,cGMP as second messengers)
 Components of co-enzymes
(NAD+, NADP+, FMN, FAD and coenzyme A)

 Messenger RNA (mRNA) directs ribosomal protein synthesis

 Transfer RNA (tRNA) transports amino acids in cell and delivers
them to the ribosome
 Ribosomal RNA (rRNA) makes up about 2/3 of the ribosome –
structural and functional roles
 Ribozymes have catalytic properties – function like an enzyme

 DNA stores the genetic code.

G
Structures of Nucleotides- (D(R)NA Building
Blocks
Without the phosphoryl group,
this would be called a nucleoside.

-D-ribofuranose

Nitrogenous bases + Pentose = Nucleosides

Nucleoside + Pi = Nuclotides.
In RNA, the sugar is ribose (OH),
in DNA, the sugar is deoxyribose (H).
DNA contains thymidine while RNA contains uracil.
*Note: the only difference is the methyl group
 Nomenclature
 A nucleoside is shown in pink,
 the nucleotide is the entire structure with at least one phosphate.
Fig . Purine deoxyribonucleotide
Fig. Pyrmidine deoxyribonucleotide
Nomenclature for components of RNA

Nomenclature for components of DNA

Biosynthesis & Degradation of nucleotides
Roles of nucleotides in metabolism
1. Nucleotides are precursors of nucleic acids and are needed for:
 replication of the genome
 transcription of the genetic material

2. ATP is the currency of energy for most biochemical pathways.

 GTP also serves as an energy source for a certain biological processes.

3. Nucleotide derivatives
 UDP-glucose participate in biosynthetic processes ~ the formation of
glycogen.

4. Nucleotides are essential components of signal-transduction

pathways.
• cAMP and cGMP are second messengers that transmit signals both within and
between cells.

5. ATP acts as the donor of phosphoryl groups transferred by

protein kinases.
 …cont’d
Purines and pyrimidines are required for:
synthesizing nucleotides and nucleic acids.

Dietary uptake of purine and pyrimidine bases is low,

b/c most of the ingested nucleic acids are
metabolized by the intestinal epithelial cells.

Biosynthetic pathways for nucleotides are of two

types:

de novo pathways

salvage pathways
 …cont’d
 In de novo (from scratch) pathways:

 the nucleotide bases are assembled from simpler

compounds.

 The framework for a purine base is synthesized piece by

piece directly onto a ribose-based structure.

 The framework for a pyrimidine base is assembled first

and then attached to ribose.

 In salvage pathways,
 preformed bases are recovered and reconnected to a
ribose moiety.
 Precursors for DNA and RNA
Both de novo and salvage pathways lead to the
synthesis of ribonucleotides.

The deoxyribonucleotides used to build DNA are

synthesized from the corresponding ribonucleotides.

The deoxyribose sugar is generated by

the reduction of ribose within a fully formed
nucleotide.

The methyl group that distinguishes the thymine of

DNA from the uracil of RNA is added at the last step
in the pathway.
 Can Cells Synthesize Nucleotides?
• Nearly all organisms synthesize purines and
pyrimidines "de novo biosynthesis pathway"
• Many organisms also "salvage" purines and
pyrimidines from diet and degradative
pathways
• Ribose generates energy, but purine and
pyrimidine rings do not.

• Nucleotide synthesis pathways are good targets

for anti-cancer/antibacterial strategies.
 Purine Biosynthesis
The purine bases are produced by de novo by pathways:
 uses amino acids, CO2, and N10_formyltetrahydrofolate as
precursors to produce nucleotides.

Most de novo synthesis occurs

 in the liver, and
 the nitrogenous bases and nucleosides are then transported to
other tissues by red blood cells.

 The brain also synthesizes significant amounts of

nucleotides.

 B/c de novo pathway requires:

 More than six high-energy bonds per purine produced.
 a salvage pathway, which is used by many cell types,
 How Do Cells Synthesize Purines?
John Buchanan (1948) "traced" the sources of all
nine atoms of purine ring
• N-1: aspartic acid
• N-3, N-9: glutamine
• C-2, C-8: N10-formyl-THF - one carbon units
• C-4, C-5, N-7: glycine
• C-6: CO2
Figure 1
The de novo pathway for purine
synthesis.
Step 1: Ribose-5-phosphate
pyrophosphokinase.
Step 2: Glutamine phosphoribosyl
pyrophosphate amidotransferase.
Step 3: Glycinamide ribonucleotide (GAR)
synthetase.
Step 4: GAR transformylase.
Step 5: FGAM synthetase (FGAR
amidotransferase).
Step 6: FGAM cyclase (AIR synthetase).
Step 7: AIR carboxylase.
Step 8: SAICAR synthetase.
Step 9: adenylosuccinase.
Step 10: AICAR transformylase.
Step 11: IMP synthase.
 IMP Biosynthesis
The first purine product of this pathway, IMP
(inosinic acid or inosine monophosphate)
• First step: Ribose-5-phosphate pyrophosphokinase
– PRPP synthesis from ribose-5-phosphate and ATP
– PRPP is limiting substance for purine synthesis
– But PRPP is a branch point so next step is the
committed step (fig 1)
• Second step: Gln PRPP amidotransferase
– Form phosphoribosyl--amine; Changes C-1
configuration (→)
– GMP and AMP inhibit this step - but at distinct sites
– Azaserine - Gln analog - inhibitor/anti-tumor
Figure 2 The structure of azaserine.
Azaserine acts as an irreversible inhibitor of glutamine-
dependent enzymes by covalently attaching to nucleophilic
groups in the glutamine-binding site.
• Step 5: Formylglycinamide ribonucleotide
(FGAR) amidotransferase
– Irreversibly inactivated by azaserine

• 6 ATPs, but that this is really 7 ATP

equivalents

• The dependence of purine biosynthesis on

THF in two steps means that methotrexate
and sulfonamides block purine synthesis
 Folate Analogs as Antimicrobial and
Anticancer Agents
• De novo purine biosynthesis depends on folic acid
compounds at steps 4 and 10
• For this reason, antagonists of folic acid metabolism
indirectly inhibit purine formation and, in turn, nucleic
acid synthesis, cell growth, and cell development.

• Rapidly growing cells, such as infective bacteria and fast-

growing tumors, are more susceptible to such agents.
• Sulfonamides are effective anti-bacterial agents
• Methotrexate and aminopterin are folic acid analogs that
have been used in cancer chemotherapy
Sulfa drugs, or sulfonamides, owe their antibiotic properties to their similarity to p-
aminobenzoate (PABA),an important precursor in folic acid synthesis.
Sulfonamides block folic acid formation by competing with PABA.
 AMP and GMP are Synthesized from IMP
Reciprocal control occurs in two ways - see
Figures 3 and 4

• GTP is the energy input for AMP synthesis,

whereas ATP is energy input for GMP
• AMP is made by N addition from aspartate (in
the familiar way - see Figure 3)

• GMP is made by oxidation at C-2, followed by

replacement of the O by N (from Gln)
• Last step of GMP synthesis is identical to the
first two steps of IMP synthesis
Figure 3 The synthesis of AMP
and GMP from IMP.
Starting from ribose-5-phosphate
8 ATP equivalents are consumed in
the AMP synthesis
9 ATP equivalents in GMP
synthesis
 The regulation of purine synthesis
Reciprocal control occurs in two ways
IMP synthesis:
– Allosterically regulated at the first two steps
1. R-5-P pyrophosphokinase:
• ADP & GDP
2. Gln-phosphoribosyl pyrophosphate
amidotransferase
• A “series”: AMP, ADP, and ATP
• G “series”: GMP, GDP, and GTP
• PRPP is “feed-forward” activator
AMP synthesis:
Adenylosuccinate synthetase is feedback-inhibited by
AMP
GMP synthesis:
IMP dehydrogenase is feedback-inhibited by GMP

Fig 4
 Nucleoside diphosphate and
triphosphate
Nucleoside diphosphate: ATP-dependent kinase
– Adenylate kinase: AMP +ATP → ADP +ADP
– Guanylate kinase: GMP +ATP → GDP +ADP

Nucleotide triphosphate: non-specific enzyme

– Nucleoside diphosphate kinase
GDP +ATP  GTP +ADP
NDP +ATP  NTP +ADP (N=G, C, U, and T)
 Can Cells Salvage Purines?
• Salvage pathways
– Recover them in useful form
– Collect hypoxanthine and guanine and recombine
them with PRPP to form nucleotides in the
HGPRT reaction
– Absence of HGPRT is cause of Lesch-Nyhan
syndrome (sex-linked); In Lesch-Nyhan, purine
synthesis is increased 200-fold and uric acid is
elevated in blood
• HGPRT
 Hyperxanthine-Guanine PhosphoRibosylTransferase

Figure 5 Purine salvage by the HGPRT reaction.

Victims of Lesch-Nyhan syndrome
experience severe arthritis due to
accumulation of uric acid, as well as
retardation, and other neurological
symptoms.

Lesch-Nyhan syndrome
results from a complete
deficiency in HGPRT.
 How Are Purines Degraded?
Purine catabolism leads to uric acid
• Nucleotidases and nucleosidases release ribose and
phosphates and leave free bases
– Nucleotidase: NMP + H2O → nucleoside + Pi
– Nucleosidase: nucleoside + H2O → base + ribose
– PNP: nucleoside + Pi → base + ribose-P
• The PNP products are converted to xanthine by
xanthine oxidase and guanine deaminase
• Xanthine oxidase converts xanthine to uric acid
– Note that xanthine oxidase can oxidize two different sites
on the purine ring system

• Neither adenosine nor deoxyadenosine is a substrate

for PNP
–
In mammals other than higher
primates, uricase converts uric
acid to the water-soluble product
allantoin.
However, since humans lack
uricase, the end product of purine
catabolism in humans is uric acid.

Figure 6. The major pathways

for purine catabolism in animals.
Catabolism of the different
purine nucleotides converges in
the formation of uric acid.
Severe combined immunodeficiency syndrome (SCID)

The effect of elevated levels of deoxyadenosine on purine metabolism.

If ADA is deficient or absent, deoxyadenosine is not converted into deoxyinosine as
normal .
Instead, it is salvaged by a nucleoside kinase, which converts it to dAMP, leading to
accumulation of dATP and inhibition of deoxynucleotide synthesis . Thus, DNA
replication is stalled.
 Xanthine Oxidase and Gout
• Xanthine Oxidase in liver, intestines mucosa,
and milk can oxidize hypoxanthine to xanthine
and xanthine to uric acid
• Humans and other primates excrete uric acid in
the urine, but most N goes out as urea
• Birds, reptiles and insects excrete uric acid and
for them it is the major nitrogen excretory
compound
• Gout occurs from accumulation of uric acid
crystals in the extremities
• Allopurinol, which inhibits xanthine oxidase ,
is a treatment
 Figure 8 Allopurinol, an
analog of hypoxanthine, is a
Figure 7 Xanthine oxidase catalyzes a potent inhibitor of xanthine
hydroxylase-type reaction. oxidase.
How Do Cells Synthesize Pyrimidines?
• In contrast to purines, pyrimidines are not
synthesized as nucleotides
– The pyrimidine ring is completed before a ribose-
5-P is added
• Carbamoyl-P and aspartate are the precursors
of the six atoms of the pyrimidine ring
Figure 9
The de novo pyrimidine biosynthetic pathway.
 UTP and CTP
• Nucleoside monophosphate kinase
UMP + ATP → UDP + ADP
• Nucleoside diphosphate kinase
UDP + ATP → UTP + ADP
• CTP sythetase forms CTP from UTP and ATP
Regulation of pyrimidine biosynthesis
• In bacteria
– allosterically inhibited at ATCase by CTP (or
UTP)
– allosterically activated at ATCase by ATP
(compete with CTP)

• In animals
– UDP and UTP are feedback inhibitors of CPS II
– PRPP and ATP are allosteric activators
Figure 10
A comparison of the regulatory circuits that control pyrimidine synthesis in E. coli and
animals.
 How Are Pyrimidines Degraded?
• In humans, pyrimidines are recycled from
nucleosides (via phosphoribosyltransferase),
but free pyrimidine bases are not salvaged
• Catabolism of cytosine and uracil yields -
alanine, ammonium, and CO2
 -alanine can be recycled into the synthesis of
coenzyme A
• Catabolism of thymine yields -
aminoisobutyric acid, ammonium, and CO2
Figure 11 Pyrimidine degradation.
Carbons 4, 5, and 6 plus N-1 are released
as -alanine, N-3 as NH4+, and C-2 as
CO2. (The pyrimidine thymine yields -
aminoisobutyric acid.)
Recall that aspartate was the source of N-
1 and C-4, -5, and -6, while C-2 came
from CO2 and N-3 from NH4+ via
glutamine.
 How Do Cells Form the
Deoxyribonucleotides That Are
Necessary for DNA Synthesis?
• Reduction at 2’-position of ribose ring
• Serve as precursor for DNA synthesis
• Replacement of 2’-OH with hydride is catalyzed
by ribonucleotide reductase
– An 22-type enzyme - subunits R1 (86 kD) and R2
(43.5 kD)
– R1 has two regulatory sites, a specificity site and an
overall activity site
Figure 12
Deoxyribonucleotide
synthesis involves
reduction at the 2'-position
of the ribose ring of
nucleoside diphosphates.
 Regulation of dNTP Synthesis
• The overall activity of ribonucleotide
reductase must be regulated
– ATP activates, dATP inhibits at the overall
activity site

• Balance of the four deoxynucleotides must

be controlled
– ATP, dATP, dTTP and dGTP bind at the
specificity site to regulate the selection of
substrates and the products made
 How Are Thymine Nucleotides
Synthesized?
• Thymine nucleotides are made from dUMP, which
derives from dUDP, dCDP

• Thymidylate synthase methylates dUMP at 5-

position to make dTMP
• N5,N10-methylene THF is 1-C donor
• If the dCDP pathway is traced from the common
pyrimidine precursor, UMP, it will proceed as
follows:
UMP  UDP  UTP  CTP  CDP  dCDP  dCMP  dUMP  dTMP
Figure 13 (a) The dCMP deaminase reaction.
An alternative route to dUMP is provided by dCDP, which is dephosphorylated
to dCMP and then deaminated by dCMP deaminase
• Synthesis of dTMP from dUMP is catalyzed by
thymidylate synthase.

• This enzyme methylates dUMP at the 5-position to

create dTMP

• The methyl donor is the one-carbon folic acid

derivative N5,N10-methylene-THF

• The reaction is a reductive methylation; the one-

carbon unit is transferred at the methylene level of
reduction and then reduced to the methyl level
Figure 14
The thymidylate
synthase reaction.
Precursors and analogs
of folic acid employed
as antimetabolites:
sulfonamides , as well
as methotrexate,
aminopterin, and
trimethoprim, whose
structures are shown
here.
These compounds
shown here bind to
dihydrofolate reductase
(DHFR) with about
1000-fold greater
affinity than DHF and
thus act as virtually
irreversible inhibitors.