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
• Decreased IMP and GMP
Increased de novo
purine synthesis
 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 , 25 th ed. C. 2000.
Appleton and lange. Pp386-401
THANK YOU!!!