NUCLEOTIDE

METABOLISM
By:
Rebecca Asis Villanueva M.D.
Associate Professor
Department of Biochemistry & Nutrition
 NUCLEOTIDE
 Ribonucleoside and deoxyribonucleoside
phosphate
 Essential for all cells
 Serve as carriers of activated intermediates in
the synthesis of some carbohydrates, lipids
and proteins
 Structural component of:
coenzyme A
FAD
NAD
NADP
 Important regulatory compounds for
many pathways of intermediary
metabolism inhibiting or activating
key enzymes.
 “energy currency” in the cell
 Nucleotide structure
 Composed of nitrogenous base, a
pentose monosaccharide, and one
two or three phosphate groups.
 Nitrogen containing bases are:
purine
pyrimidine
Purine Structure

Guanine
Pyrimidine structure

cytosine
Pyrimidine structure

thymine
Pyrimidine structure

Uracil
•DNA and RNA contain:
purine bases: adenosine and guanine
pyrimidine base: cytosine

•DNA contains thymine as second pyrimidine base

•RNA contains Uracil as second pyrimidine base

•Thymine and Uracil differ only by one methyl

group present in thymine but absent on Uracil

i. e. some viral DNA and transfer RNA

Base modification:

methylation
hydroxymethylation
glycosylation
acetylation
alterations of the atoms in pyrimidine
ring
•Presence of unusual base in a nucleotide
sequence:
-aid in its recognition by specific
enzyme
- protect it from being degraded by
nucleases
 Nucleosides
 Addition of pentose sugar to a base
produces a nucleoside.
Adenine Adenosine
Guanine Guanosine
Cytosine Cytidine
Thymine Thymidine
Uracil Uridine
•Sugar:
ribose ribonucleoside
2-deoxyribose deoxyribonucleoside

Carbon and nitrogen atoms in the rings of the

base and the sugar are numbered separately

Atoms in the rings of the bases are numbered

1-6 in pyrimidine and 1-9 in purines

Carbons in the pentose are numbered 1-5

 Nucleotides
 Mono, di or triphosphate esters of
nucleosides
 Phosphate group attached by linkage
to the 5’-OH of the pentose is called
nucleoside 5’-phosphate or
5’nucleotide
 Type of pentose denoted prefix in the
names “5’ ribonucleotide and 5’
deoxyribonucleotide”
•Phosphate group are responsible for the
negative changes associated with nucleotides
and nucleic acids

•If phosphate group is attached to the 5

carbon of the pentose -Nucleoside
monophosphate (NMP)
i.e. AMP and CMP
•If second or third phosphate is added to the
nucleoside - nucleoside diphosphate,
triphosphate
i.e. ADP and ATP
 Biomedical Importance
 Biosynthesis of purines and pyrimidines
are regulated and coordinated by
feedback mechanism
 Production in quantities vary in times,
which maybe appropriated with
physiologic demands
 Genetic diseases associated with
purine metabolism

 Gout
 Lesch-Nyhan syndrome
 Adenosine deaminase deficiency
 Purine nucleoside phosphorylase
deficiency
 Diseases associated with
pyrimidine catabolism

 Few clinically significant disorders

 Orotic acidemias
 Purine and pyrimidine are dietarily
nonessential
 Human tissue synthesize purine and
pyrimidine from amphibolic
intermediates
 Ingested nucleic acids and
nucleotides
 Dietarily nonessential
 Degraded in the intestinal tract to
monosaccharide which may be
absorbed or converted to purine and
pyrimidine bases.
 Purine base
 Converted to uric acid by oxidation
 May be absorbed and excreted in the
urine
DE NOVO PURINE
NUCLEOTIDE
SYNTHESIS
 Atoms of the purine ring are
contributed by:
-amino acids
-CO2
-derivatives of tetrahydrofolate
Sources of Individual Atoms in
Purine Ring

N5
N

N
 Synthesis of 5-phosphoribosyl-1-
pyrophosphate
 Ribose phosphate
pyrophosphokinase (PRPP
synthetase) is activated by Pi and
inhibited by purine nucleoside di and
triphosphates.
 Synthesis of 5’-
phosphoribosylamine
 The amide of glutamine replaces the
pyrophosphate group attached to
carbon 1 of PRPP. The enzyme
glutamine: phosphoribosyl
pyrophosphate amidotransferase is
inhibited by the purine 5’-nucleotides
AMP,GMP and IMP, the end products
of this pathway. This is the
committed step in purine nucleotide
biosynthesis
 Synthesis of inosine
monophosphate, the “parent”
purine nucleotide
-Requires 4 ATP molecules as an

energy source.
 Inhibitors of purine synthesis
 specific for inhibiting the growth of
rapidly dividing microorganisms( ex.
Sulfonamides )
 Structural analog s of folic acid (ex.
Methotrexate) are use
pharmacologically to control the
spread of cancer by interfering the
synthesis of nucleotides, and
therefore DNA and RNA
 Conversion of IMP to AMP and
GMP
 The conversion of IMP to either AMP
or GMP utilizes a two-step, energy
requiring pathway. Note that the
synthesis of AMP requires GTP as an
energy source, whereas the
synthesis of GMP requires ATP
 the first reaction in each pathway is
inhibited by the end product of that
pathway. This provides a mechanism
for diverting IMP to the synthesis of
the species of purine present in
lesser amounts.
 Conversion of nucleoside
monophosphates to nucleoside di
and triphosphates
 Nucleoside diphosphates are
synthesized from the corresponding
nucleoside monophosphates by base-
specific nucleoside monophosphate
kinases.
 ATP is generally the source of the
transferred phosphate because it is
present in higher concentrations than
the other nucleoside triphosphates.
 For example, adenylate kinase:
AMP + ATP ↔ 2 ADP
 For example, guanylate kinase:
GMP + ATP ↔GDP + ADP
 Adenylate kinase is particularly
active in liver and muscle, where the
turnover of energy from ATP is high.
Its function is to maintain an
equilibrium among AMP,ADP, and
ATP:
 2ADP ↔ AMP + ATP
 Nucleoside diphosphates and
triphosphates are interconverted by
nucleoside diphosphate kinase- an
enzyme that, unlike the
monophosphate kinases, had broad
specificity.
 For example,
 GDP + ATP ↔ GTP + ADP
 For example,
 CDP + ATP ↔ CTP + ADP
 SALVAGE PATHWAY FOR
PURINE
 Purines from the normal turnover of
cellular nucleic acids or obtained
from diet that are not degraded can
be reconverted into nucleoside
triphosphate
 Energetically much less expensive
than complete de novo synthesis
 Deficiency causes Lesch-Nyhan
syndrome
 Enzymes Involved in Salvage
Pathway
 Adenine phosphoribosyl transferase
 Hypoxanthine-guanine
phosphoribosyl transferase

 Both utilize PRPP as source of ribose-5-

phosphate group
 Release of pyrophosphate makes these
reactions irreversible
 Salvage Pathway
Two key transferase enzymes are involved in the salvage of
purines: adenosine phosphoribosyltransferase (APRT), which
catalyzes the following reaction:

adenine + PRPP <-----> AMP + PPi

and hypoxanthine-guanine phosphoribosyltransferase (HGPRT),
which catalyzes the following reactions:

hypoxanthine + PRPP <------> IMP +

PPi
guanine + PRPP <--------> GMP + PPi
 Lesch-Nyhan Syndrome
 Complete deficiency of
hypoxanthine-guanine
phosphoribosyl transferase
 Inability to salvage hypoxanthine or
guanine
 Excessive production of uric acid
 Increased levels of PRPP Increased de novo
purine synthesis
 Decreased IMP and GMP
 DEGRADATION OF PURINE
NUCLEOTIDES
 Degraded by removal or alterations
of portions of the nucleotide
 End product in human: Uric Acid
 Other mammals oxidize uric acid to allantoin
which can further be degraged to urea or
ammonia
Formation of Uric Acid
 Degradation of Dietary Nucleic
Acids in Small Intestine
DNA / RNA
mouth

Low pH denatures DNA & RNA

stomach

Denatured nucleic acids

nucleases
from pancreas

Oligonucleotides
small intestine
Purines / Primidines
phosphodiesterases
from pancreas

Mononucleotides nucleotidases Nucleosides nucleosidases

 PYRIMIDINE SYNTHESIS
 Pyrimidine ring synthesized before
being attached to ribose-5-
phosphate
 Sources of carbon and nitrogen
atoms:
 Glutamine
 CO2
 Aspartic acid
Pyrimidine Synthesis
Synthesis of Carbamoyl Phosphate
 Committed step
 Catalyzed by: Carbamoyl phosphate
synthetase II
 Inhibited by UTP
 Activated by ATP and PRPP
 cytosolic
 Uses γ-amide group of amine as
source of nitrogen
 Synthesis of Orotic Acid
 Second step: formation of carbamoyl
aspartate
 Catalyzed by aspartate transcarbamoylase
 Pyrimidine ring closed by
dihydroorotase resulting to
dihydroorotate oxidized to orotic
acid
Formation of Pyrimidine Nucleotide
 Complete pyrimidine ring converted
to orotidine 5’-monophosphate
 PRPP – ribose 5-phosphate donor
 Orotate phosphoribosyl transferase
produces OMP with release of
pyrophosphate
 Biologically irreversible
 OMP converted to UMP
 By Orotidylate decarboxylase
 Deficiency: Orotic aciduria
 Synthesis of Uridine Triphosphate
and Cytidine Triphosphate
 Amination of UTP
 By CTP synthase
 Nitrogen provided
by glutamine

Synthesis of CTP from UTP

 DEGRADATION OF PYRIMIDINE
NUCLEOTIDES
 Pyrimidine rings can be degraded to
highly soluble structures
 Such as β- alanine & β-aminoisobutyrate
 Can be salvaged and converted into
nucleotides
 By pyrimidine phosphoribosyltransferase
 Utilizes PRPP as source of ribose-P
CONVERSION OF RIBONUCLEOTIDES TO
DEOXYRIBONUCLEOTIDES

RIBONUCLEOTIDES

Use as building blocks in RNA synthesis.

as nucleotide carriers of other compound

The nucleotides required for DNA synthesis are

2’ – deoxyribonucleotides, which are produced from
ribonucleoside diphosphate
 A. RIBONUCLEOTIDE REDUCTASE

 Ribonucleotide reductase (ribonucleoside

diphosphate reductase )
 A multi subunit enzyme ( two identical B1
subunits and two identical B2 subunits) that is
specific for reduction of nucleoside diphosphates
(dADP,dGDP,dCDP, and dUDP )
 Immediate donors of hydrogen atom needed for
the reduction of the 2’-hydroxyl group are two
sulfhydryl groups on the enzyme itself—which
during rreaction forms a disulfide bond
 CONVERSION OF RIBONUCOTIDES TO
DEOXYRIBONUCLEOTIDES

O O
BASE O O
O-P-O-P-P-CH2 O O BASE
O-P-O-P-O-CH2
O O H
H O O H
H H H
H H
OH OH OH H
RIBONUCLEOSIDE DEOXYRIBONUCLEOSIDE
DIPHOSPHATE DIPHOSPHATE

Ribonucleotide diphosphate Deoxyribonucleotide diphosphate

Ribonucleotide reductase
H20
Thioredoxin (2 SH) Thioredoxin ( S – S)
(reduced) (oxidized)
Thioredoxin reductase

NADP+ NADPH + H+
1.Regeneration of reduced enzyme :

 for fibonucleotide reductase to continue to produce deoxy

Ribonucleotides, the disulfide bong created during production of the
2’-deoxy carbon must be reduced.

The source of reducting equivalents is a peptide co enzyme of

Ribonucleotide reductase , THIOREDOXIN.

Thioredoxin – contains 2 Cysteine residues separated by two amino

acids in peptide chain.

The 2 sulfhydryl groups of thioredoxin donate their hydrogen atoms to

ribonucleotide reductase in the process of forming a disulfide bond.
2. Regeneration of reduced Thioredoxin :

 Thioredoxin must be converted back to its reduced form in

Order to continue its function.

 The necessary equivalents are provided by NADPH + H, and

the reaction is catalyzed by Thioredoxin reductase.
 B. REGULATION OF DEOXYRIBONUCLEOTIDE
SYNTHESIS
Ribonucleotide reductase – responsible for maintaining a balance supply
Of the deoxyribonuclotides required for DNA synthesis.

 to achieve this, the regulation of the enzyme is complex. In addition

to the single active site, there are two sites on the enzyme involved
In regulating this activity.

1. Activity Site
- the binding of dATP to an Allosteric site (known as the activity site),
inhibits the overall catalytic activity of the enzyme.

2. Substrate specificity site :

- the binding of nucleoside triphosphate to an additional allosteric site
(known as the substrate specificity site ) on the enzyme regulates
Substrate specificity, causing an increase in the conversion of
Ribonucleotides to deoxyribonucleotides as they are required for DNA
synthesis.
REGULATION OF RIBONUCLEOTIDE REDUCTASE
ACTIVITY SITES
SUBSTRATE SPECIFICITY SITE

B1 B1
subunit subunit

SH SH SH SH

B2 B2
Ribonucleoside
subunit subunit Deoxyribonucleoside
Diphosphate Diphosphate (dNDP)
(NDP)
 SYNTHESIS OF THYMIDINE
MONOPHOSPHATE FROM dUMP

dUMP is converted to dTMP by thymidylate synthetase, w/c

Utilizes N5, N10 – methylene tetrahydrofolate as the source of the
methyl group.

 this is unusual reaction in that tetrahydrofolate (THF) contributes

not only a carbon unit but also TWO hydrogen atoms for the
pteridine ring, resulting in the oxidation of THF to dihydrofolate
(DHF)
 Inhibitors of Thymidylate synthetase include thymine analogs
such as 5- fluoracil.

 DHF can be reduced to THF by dihyrofolate reductse, an enzyme

That is inhibited in the presence of drugs such as methotrexate.

 by decreasing the supply of THF, these folate analogs not only

Inhibit purine synthesis, but by preventing methylation of dUMP
to dTMP, they also lower the cellular concentration of this essential
Component of DNA

 DNA synthesis is therefore inhibited and cell growth slowed.

 Disorders of Purine
Catabolism:
I. GOUT (Metabolic Disorder of Purine
Catabolism)
 Various genetic defects in PRPP synthetase
present clinically as Gout.
 Results in overproduction and overexcretion of
purine catabolites or resistance to feedback
inhibition.
e.g. An elevated Vmax increased affinity for
ribose 5-phosphate.
 Gouty Arthritis
 Disorders of Purine
Catabolism:
II. Lesch- Nyhan Syndrome
 An overproduction hyperuricimia characterized by
frequent episodes of uric acid lithiasis and a bizarre
syndrome of self mutilation
 The accompanying rise in intracellular PRPP results
in purine overproduction.
 Mutations that decrease or abolish hypoxanthine-
guanine phosphoribosyltransferase activity include
deletions, frameshift mutations, base substitutions,
and aberrant mRNA splicing.
 Disorders of Purine
Catabolism:
III. Von Gierke’s Disease
 Purine overproduction and hyperuricemia
(glucose-6-phosphatase deficiency) occurs
secondary to enhanced generation of the PRPP
precursor ribose 5-phosphate.

IV. Hypouricemia
 Hypouricaemia and increased excretion of
hypoxanthine and xanthine are associated
with xanthine oxidase deficiency due to
genetic defect or to severe liver damage.
 Disorders of Purine
Catabolism:
V. Adenosine Deaminase & Purine
Nucleoside Posphorylase Deficiency
 A deficiency which is associated with an
immunodeficiency disease in which both thymus
derived lymphocytes (T cells) and bone marrow-
derived lymphocytes (B cells) are spurse and
dysfunctional.
 Purine nucleoside phosphorylase deficiency is
associated with a severe deficiency of T cells but
apparently normal B cell function.
 Immune dysfunctions appear to result from
accumulation of dGTP and dATP, which inhibit
ribonucleotide reductase and thereby deplete cells of
Overproduction of Pyrimidine Catabolites
is only rarely associated with clinically
significant abnormalities
 Since the end products of pyrimidine catabolism
are highly water-soluble, pyrimidine
overproduction results in few clinical signs or
symptoms.

 In hyperuricemia associated with severe

overproduction of PRPP, there is overproduction of
pyrimidine nucleotides and increased excretion of
ß-alanine. Since N5, N10 –methylene-
tetrahydrofolate is required for thymidylate
synthesis, disorders of folate and vitamine B12
metabolism results in deficiencies of TMP.
 Clinically Significant
Abnormalities
I. Orotic Acidurias
 The orotic acidurias that accompanies Reye’s
syndrome probably is consequence of the inability
of severely damaged mitochondria to utilize
carbamoyl phosphate which then becomes
available for cytosolic overproduction of orotic
acid.
 Type I orotic aciduria reflects a deficiency of
both orotate phosphoribosyltransferase and
orotidylate decarboxylase.
 The rarer Type II orotic aciduria is due to a
deficiency only of orotidylate decarboxylase.
 Clinically Significant
Abnormalities
II. Deficiency of a Urea Cycle Enzyme
results in Excretion of Pyrimidine
Precursors
 Increased excretion of orotic acid, uracil, and
uridine accompanies a deficiency in liver
mitochondrial ornithine transcarbamoylase.
 Excess carbamoyl phosphate exits to the
cytosol, where it stimulates pyrimidine
nucleotide biosynthesis.
 The resulting mild orotic aciduria is increased
by high nitrogen containing food.
 Clinically Significant
Abnormalities
III. Drugs May Precipitate Orotic
Aciduria
 Allopurinol, an alternative substrate for
orotate phosphoribosyltransferase, competes
with orotic acid.
 The resulting nucleotide product also inhibits
orotidylate decarboxylase, resulting in orotic
aciduria and ortidinuria.
 6-Azauridine, following conversion to 6-
azauridylate, also competitively inhibits
orotidylate decarboxylase, enhancing
exretion of orotic acid and orotidine
 INHIBITORS OF PURINE METABOLISM
(CANCER CHEMOTHERAPY)
III. The turnover of nucleic acids in malignant of its formation
in these cells may offer a therapeutic value. Since most if
not. All of these “inhibitor drugs” are non-specific, then
there is a possibility that it can also affect normal tissues
which are rapidly undergoing mitotic cycles like the
hematopoietic tissues hence should be administered with
proper caution.
1. 6-Mercaptopurine (6MP)
This is an antitumor drug which is an analougue of adenine
metabolized to a ribonucleotide by the APRT salvage
pathway thus inhibiting the conversion of IMP to GMP or
AMP. It also inhibits the rate limiting step of the purine
de novo pathway. Simultaneous administration with
allopurinol potentiates its effect as 6-Mercaptopurine will
not be degraded and therefore will delay its inactivation.
 INHIBITORS OF PURINE METABOLISM
(CANCER CHEMOTHERAPY)

2. Adenosine arabinoside (the sugar is replaced by

an arabinose)
This is used as an antiviral and an antitumor drug in
man. It inhibits DNA polymerase after its
conversion to the triphosphate form.
3. Azaserine
An analogue of glutamine, thus inhibits the
incorporation of N3 and N9 into the purine ring
(de novo biosynthesis), inhibits formation of
GMP from IMP, CTP from UTP-all reactions
requiring the entry of glutamine.
 INHIBITORS TO PYRIMIDINE NUCLEOTIDE
METABOLISM (CANCER AND VIRAL
CHEMOTHERAPY)
1. Aminopterine/Amithopterine/Methotrexate
-Inhibits dihydrofolate reductase.
2. 5 Flurouracil (5 FU)
- An analogue of thymine used in the treatment of solid
tumors. It is converted to the monophosphate nucleotide
form via the salvage pathway. Eventually it is converted
to the deoxynucleotide form and binds to thymidylate
synthetase, thereby inhibiting the formation of TMP. As a
deoxytriphosphate form, it can be incorporated into RNA
and inhibits the formation of mature RNA (a step
important in translation)
3. 5-Iodouracil
- Functions as an analogue of thymidine, which when
incorporated into DNA bonds with C rather than A, thus
causing misreading of the strand.
 INHIBITORS TO PYRIMIDINE NUCLEOTIDE
METABOLISM (CANCER AND VIRAL
CHEMOTHERAPY)
4. Cytosine arabinoside (sugar is replaced to an arabinose)
-used in the treatment of leukemias. It ingibits DNA
polymerase in its triphosphate form. It has a short half
life.

REFERENCES:
Daubner, SC et al. Biochemistry. 24:7059-7065

Lee L. et al. oligomeric structure of the multifunctional protein

CAD that initiates pyrimidine biosynthesis in
mammalian cells. Proc. Nat. Acad of Sci. 1985,
82:6802-6806.
Murray, Robert K, et al. Harper’s review of biochemistry , 25th
ed. C. 2000. Appleton and lange. Pp386-401
THANK YOU!!!