Nucleotides chemistry and metabolism

NUCLEOTIDES:
CHEMISTRY AND METABOLISM

Nucleotides:
Chemistry and Metabolism
Chapter at a Glance
Purines and pyrimidines
Nucleosides and nucleotides
De novo synthesis of purine nucleotides
Degradation of purine nucleotides
Uric acid and gout
De novo synthesis of pyrimidines
Disorders of pyrimidine metabolism

Nucleotides
In 1868, Friederich Miescher isolated nucleic acid (then
called nuclein) from pus cells.
 Albrecht Kossel (Nobel prize, 1910) differentiated RNA
and DNA in 1882.
 In 1906, Kossel described the 4 bases in nucleic acids.
Nucleotides are precursors of the nucleic acids,
deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA).
 The nucleic acids are concerned with the storage and
transfer of genetic information.
The universal currency of energy, namely ATP, is a
nucleotide derivative.
Nucleotides are also components of important co-
enzymes like NAD+ and FAD, and metabolic regulators
such as cAMP and cGMP.

Composition of Nucleotides
A nucleotide is made up of 3 components:
a. Nitrogenous base (a purine or a pyrimidine)
b. Pentose sugar, either ribose or deoxyribose
c. Phosphate groups esterified to the sugar.
When a base combines with a pentose sugar, a
nucleoside is formed.

Composition of Nucleotides
When the nucleoside is esterified to a phosphate
group, it is called a nucleotide or nucleoside
monophosphate.
When a second phosphate gets esterified to the
existing phosphate group, a nucleoside diphosphate
is generated.
The attachment of a 3rd phosphate group results in
the formation of a nucleoside triphosphate.
 The nucleic acids (DNA and RNA) are polymers of
nucleoside monophosphates.

Bases Present in the Nucleic Acids
 Two types of nitrogenous bases; the purines and pyrimidines are
present in nucleic acids.
Purine Bases
 The purine bases present in RNA and DNA are the same; adenine
and guanine.
 Adenine is 6-amino purine and guanine is 2-amino, 6-oxopurine.
 The numbering of the purine ring with the structure of adenine
and guanine are shown in Fig.
Minor Purine Bases
 These are hypoxanthine (6-oxopurine) and xanthine (2, 6-di-
oxopurine)
 Uric acid (2,6,8-tri-oxopurine) is formed as the end product of the
catabolism of other purine bases.
 It can exist in the "enol" as well as "keto" forms (tautomeric forms)
 Keto form is by far the predominant type under physiological
conditions.

Bases Present in the Nucleic Acids
• Pyrimidine Bases
 The pyrimidine bases present in nucleic acids are cytosine, thymine
and uracil.
 Cytosine is present in both DNA andRNA. Thymine is present in
DNA and uracil in RNA.
 A few other modified pyrimidine bases like dihydrouracil and 5-
methyl cytosine are also found rarely in some types of RNA.

Nucleosides
i. Nucleosides are formed when bases are attached to
the pentose sugar, D-ribose or 2-deoxy-D-ribose.
ii. All the bases are attached to the corresponding
pentose sugar by a beta-N-glycosidic bond between
the 1st carbon of the pentose sugar and N9 of a
purine or N1 of a pyrimidine.
iii. The deoxy nucleosides are denoted by adding the
prefix d- before the nucleoside.
iv. The carbon atoms of the pentose sugar are
denoted by
using a prime number to avoid confusion with the
carbon atoms of the purine or pyrimidine ring

Nucleosides
The names of the different nucleosides are given in
v. Nucleosides with purine bases have the suffix -sine,
while pyrimidine nucleosides end with -dine.
vi. Uracil combines with ribose only; and thymine with
deoxy ribose only .
v. Nucleosides with purine bases have the suffix –sine
, while pyrimidine nucleosides end with -dine.
vi. Uracil combines with ribose only; and thymine with
deoxy ribose only.

Nucleotides
 i. These are phosphate esters of nucleosides.
 Base plus pentose sugar plus phosphoric acid is a nucleotide.
ii. The esterification occurs at the 5th or 3rd hydroxyl group of the pentose
sugar.
 Most of the nucleoside phosphates involved in biological function are 5'-
phosphates .
iii. Since 5'-nucleotides are more often seen, they are simply written without
any prefix.
 For example, 5'- AMP is abbreviated as AMP; but 3' variety is always
written as 3'-AMP.
iv. Moreover, a base can combine with either ribose or deoxy ribose, which in
turn can be phosphorylated at 3' or 5' positions.
v. Many co-enzymes are derivatives of adenosine monophosphate ( NAD+,
NADP, FAD and Co-enzyme A) .
vi. Nucleotides and nucleic acids absorb light at a wavelength of 260 nm; this
aspect is used to quantitate them.
 As nucleic acids absorb ultraviolet light, chemical modifications are
produced leading to mutation and carcinogenesis.

Nucleoside Triphosphates
i. Corresponding nucleoside di- and triphosphates
are formed by esterification of further phosphate
groups to the existing ones.
ii. In general, any nucleoside triphosphate is
abbreviated as NTP or d-NTP .
ii. Nucleoside diphosphate contains one high energy
bond and triphosphates have 2 high energy bonds.
ATP is the universal energy currency

Nucleoside Triphosphates
 It is formed during oxidative processes by trapping the
released energy in the high energy phosphate bond.
iii. A phosphodiester linkage may be formed between the 3' and
5' positions of ribose group-called cyclic nucleotides .
 3', 5'-cyclic AMP or cAMP is a major metabolic regulator.
 Cyclic GMP also behaves similarly.
 These are second messengers in mediating the action of
several hormones.
iv. Deoxy ribonucleotides are used for synthesis of DNA and
ribonucleotides for RNA.
 In pseudouridylic acid (found in tRNA) uridine is attached to
ribose phosphate in a C-C bond instead of C-N bond in UMP.
 Active methionine, amino acid adenylates, active sulfate, etc.
are higher energy compounds containing adenosine
monophosphate.

Digestion of Nucleic Acids
The nucleic acids in the diet are hydrolyzed to a mixture
of nucleotides by ribonuclease and deoxy ribonuclease
present in pancreatic and intestinal secretions.
Then nucleotidases liberate the phosphate from
nucleotides.
The resulting nucleosides are hydrolyzed by
nucleotidases forming free bases and pentose sugars.
However, the dietary purines and pyrimidines are
neither converted to nucleotides nor incorporated into
nucleic acids.
They are directly catabolized.

BIOSYNTHESIS OF
PURINE
NUCLEOTIDES

BIOSYNTHESIS OF PURINE NUCLEOTIDES
i. The purine nucleotides are synthesized by most of
the tissues.
 However the major site is the liver.
 This pathway operates in the cytoplasm.
ii. The major pathway is denoted as de novo
synthesis, because the purine ring is synthesized
from different small components.
Since the human being can synthesize the purine
and pyrimidine bases de novo, they are said to be
prototrophs.

BIOSYNTHESIS OF PURINE NUCLEOTIDES
iv. During de novo synthesis, purine ring is built up on
a ribose-5-phosphate molecule.
 Hence, nucleotides are the products of the de novo
synthesis.
v. There are ten steps in the de novo synthesis
pathway.
The enzymes catalyzing these reactions are existing
as a multienzyme complex in eukaryotic cells.
 This arrangement increases the efficiency of the
pathway.

The names of the enzymes catalyzing the
purine synthesis steps are

The names of the enzymes catalyzing the purine synthesis
steps are
 Enzyme 1 = Phosphoribosyl amido transferase;
 Enzyme 2 = GAR synthetase;
 Enzyme 3 = GAR transformylase;
Enzyme 4 = FGAR amido transferase;
 Enzyme 5 = Cyclase;
Enzyme 6 = AIR-carboxylase;
 Enzyme 7 = SAICAR synthetase;
 Enzyme 8 = SAICAR lyase;
 Enzyme 9 = AICAR transformylase;
Enzyme 10 = IMP synthase;
Enzyme 11 = Adenylosuccinate synthetase and
adenylosuccinase.

Step 0 (Preparatory Step), PRPP synthesis
i. Phosphoribosyl pyrophosphate (PRPP) is the donor
of ribose-5-phosphate for de novo synthesis.
The reaction is:
Ribose-5-phosphate + ATP → ADP + Phosphoribosyl
pyrophosphate (PRPP).
ii. The purine ring is later on assembled on the ribose-5-
phosphate.
iii. PRPP is also used for the synthesis of pyrimidine
nucleotides, nucleotide co-enzymes and also for the
salvage pathway.
Hence the synthesis of PRPP is not considered as a step
in the de novo synthesis of purine nucleotides; it is
called a preliminary or preparatory step.

Formation of AMP
Steps 1 to 10 are summarized in Table .
The de novo synthesis of one molecule of purine
nucleotide requires 6 ATP.
First step is rate limiting. Step 4 is inhibited by
azaserine, an anticancer drug.
 6-mercaptopurine inhibits amination of IMP to
AMP, and therefore it is used as an anticancer drug.

Conversion of IMP to GMP
The conversion of IMP to GMP involves two steps,
first oxidation of IMP to xanthylic acid (xanthosine
monophosphate) (XMP) by an NAD+ dependent
dehydrogenase.
Then an amido transferase transfers the NH2 group
from glutamine to XMP to form GMP.
ATP is hydrolyzed to AMP level in this reaction .
Both AMP and GMP can be converted to their di-
and triphosphates.
Synthesis of one molecule of purine nucleotide
requires 6 high energy phosphates.

Salvage Pathway-purines
i. This pathway ensures the recycling of purines formed
by degradation of nucleotides.
Nucleosides and deoxy-nucleosides can also be
salvaged.
ii. PRPP is the starting material in this pathway; it is also
a substrate for de novo synthesis.
Hence these two pathways are closely inter-related.
iii. The free purines are salvaged by two different
enzymes; adenine phosphoribosyl transferase
(APRTase) and hypoxanthine guanine phosphoribosyl
transferase (HGPRTase).

Salvage Pathway-purines
iv. The pathway is of special importance in tissues
like RBCs and brain where the de novo pathway.
is not operating.
The salvage pathway economize intracellular energy
expenditure.

Regulation of Purine Synthesis
i. The committed step in de novo synthesis is the reaction catalyzed
by amido-transferase (step 1), It is inhibited by AMP and GMP.
ii. They act as allosteric modifiers.
 Binding of AMP and GMP on the enzyme converts monomeric
active form
to a dimeric inactive form.
iii. Since both AMP and GMP can bind to the same enzyme
molecule at different sites, they act synergistically.
iv. Both AMP and GMP inhibit their own formation by feedback
inhibition of adenylosuccinate synthetase and IMP dehydrogenase.
v. The formation of AMP from IMP requires GTP; similarly formation
of GMP requires ATP.
 Hence both GTP and ATP are made available in sufficient
quantities.
vi. The availability of PRPP is another important regulatory factor.
 The activity of PRPP synthetase is regulated by negative modifiers;
purine and pyrimidine nucleotides.

Analogs as Purine Synthesis Inhibitors
They act as competitive inhibitors of the naturally
occurring nucleotides.
They are utilized to synthesise DNA; such DNA becomes
functionally inactive.
 Thereby cell division is arrested.
So they are useful as anticancer drugs.
E.g.,a. Mercaptopurine inhibits the conversion of IMP to
GMP and AMP.
b. Folate antagonists (Methotrexate) would affect the
reactions involving one carbon group transfers.
c. Azaserine (diazoacetyl-L-Serine) is a glutamine
antagonist and therefore inhibits reactions involving
glutamine (steps 1 and 4).
d. Other synthetic nucleotide analogs used as anticancer
agents are 6-thio guanine and 8-aza guanine.

Degradation of Purine Nucleotides
The end product of purine nucleotide catabolism is
uric acid (urate).
This degradation is taking place mainly in the liver.
The xanthine oxidase is a metalloflavoprotein
containing FAD, molybdenum and iron.
As xanthine isoxidized to uric acid, the electrons are
transferred first to molybdenum, then to FAD, and
finally to molecular oxygen, when hydrogen
peroxide (one of the reactive oxygen species) is
produced.

Fig. 43.15: Degradation of purine nucleotides. Main pathway is in red arrows. PNP =
purine nucleoside phosphorylase; R-1-P = ribose- 1-phosphate

Species Difference
The end product of purine catabolism in human beings
is uric acid.
However, the total amount of nitrogen excreted as uric
acid is very little, because human beings are ureotelic.
 The amino nitrogen is finally excreted as urea in
mammals.
The birds, amphibians and reptiles are uricotelic
because they excrete uric acid as the major end
product of purine as well as amino acid catabolism.
 The lower primates and some other mammals have
the enzyme uricase which converts uric acid to
allantoin and the final product excreted is allantoin
which is more soluble.

CAFFEINE
Caffeine is the trimethyl derivative of xanthine.
Coffee and tea contains caffeine.
It inhibits phosphodiesterase, causes prolonged
action of cyclic AMP and increases the activity of
hormone sensitive lipase .
Caffeine enhances the effect of epinephrine on
glycogenolysis.

Formation of uric acid from purine
nucleosides

Fig. Formation of uric acid from purine nucleosides by way of the purine bases hypoxanthine,
xanthine, and guanine.

Fig.
Formation of uric acid from purine nucleosides
by way of the purine bases hypoxanthine,
xanthine, and guanine.
Purine deoxyribonucleosides are degraded by
the same catabolic pathway and enzymes, all of
which exist in the mucosa of the mammalian
gastrointestinal tract.

URIC ACID
Strecker ---uric acid in urine.
Sir Frederick Hopkins estimated uric acid.
Emil Fischer--- uric acid is derived from purine
nucleus.
Normal blood level of uric acid ranges from 2–5
mg/dL in females and 3–7 mg/dL in males.
The daily excretion varies from 500–700 mg.
 Nucleic acid content is more in non-vegetarian diet.
 Uric acid is sparingly soluble in water.
 Uric acid is an antioxidant .

GOUT
i. It is due to accumulation of urate crystals in the synovial fluid
resulting in inflammation leading to acute arthritis.
ii. At 30oC, the solubility of uric acid is lowered to 4.5 mg/dL.
 Therefore uric acid is deposited in cooler areas of the body to
cause tophi.
 Thus tophi are seen in distal joints of foot.
iii. Increased excretion of uric acid may cause deposition
of uric acid crystals in the urinary tract leading to
calculi or stone formation with renal damage.
 Gout may be either primary or secondary.
iv. With gout is an increased cellular pool of PRPP, the substrate
for the rate limiting step of de novo synthesis of purine
nucleotides.
 The resultant increase in the activity of amido transferase
leads to excess production of nucleotides resulting in
hyperuricemia.

Primary Gout
 About 10% of cases of primary gout are idiopathic.
 Primary gout may show a familial incidence (1:500 ).
 Causes of primary gout are:
A 5-phosphoribosyl Amido Transferase
 Active, but not sensitive to feedback regulation by the inhibitory nucleotides.
 This would lead to over production of purine nucleotides.
B Abnormal PRPP Synthetase
 Higher V max which leads to increased production of PRPP.
C Deficiency of Enzymes of Salvage Pathway
 Reactions which consume PRPP and produce more nucleotides will inhibit the
enzyme, e.g. salvage pathway.
 Primary gout is now considered as a disease of the elderly, related to lifestyle.
D Glucose-6-phosphatase Deficiency
 This condition is known as von Gierke's disease (glycogen storage disease, type I, .
When this enzyme is deficient, glucose-6-phosphate cannot be converted to
glucose.
 So more glucose is channeled into the pentosephosphate shunt pathway, resulting
in increased availability of ribose-5-phosphate.
 This would lead to increased formation of PRPP.

Secondary Hyperuricemia
Increased Production of Uric Acid
i. Rapidly growing malignant tissues, e.g. leukemias,
lymphomas, polycythemia.
ii. Hyperuricemia is also seen in cancer patients on
radiotherapy or chemotherapy (tumor lysis
syndrome) due to increased cellular turnover.
 Allopurinol also, to decrease uric acid
levels.
 Rasburicase (urate oxidase).
iii. Increased tissue damage due to trauma and raised
rate of catabolism as in starvation.

Secondary Hyperuricemia
• Reduced Excretion Rate
i. Renal failure
ii. Treatment with thiazide diuretics which inhibit
tubular secretion of uric acid
iii. Lactic acidosis and keto-acidosis due to
interference with tubular secretion.

Clinical Findings of Gout
Gouty attacks may be precipitated by high purine diet
and increased intake of alcohol.
Often the patients have a few drinks, go to sleep
symptomless, but are awakened during the early hours
of morning by excruciating joint pains.
Alcohol leads to accumulation of lactic acid.
The typical gouty arthritis affects the first
metatarsophalangeal joint (big toe), but other joints
may also be affected.
The joints are extremely painful.
 Synovial fluid will show birefringent urate crystals.
In chronic cases, uric acid may get deposited around
joints causing swelling (tophi) composed of sodium
urate.

Clinical Findings of Gout
The total urate pool (normal 1200 mg) is increased
to 3000 mg in gout patients without tophi.
 It may be as high as 30,000 mg in patients with
tophi.
In chronic gout, the deposition of urate crystals in
renal medulla occurs which progresses to
urolithiasis and renal damage.

Treatment Policies in Gout
i. Reduce dietary purine intake and restrict alcohol.
ii. Increase renal excretion of urate by uricosuric drugs, which
decrease the reabsorption of uric acid from kidney tubules, e.g.
probenecid.
iii. Reduce urate production by allopurinol, an analog of
hypoxanthine ,
 Allopurinol is a competitive inhibitor of xanthine .
 Xanthine and hypoxanthine are more soluble and so are excreted
more easily.
 Xanthine oxidase converts allopurinol to alloxanthine. It is a more
effective inhibitor of xanthine oxidase.(‘suicide inhibition‘).
iv. Colchicine, an anti-inflammatory agent is very useful
to arrest the arthritis in gout.
v. Use of PEG (polyethylene glycol)-uricase and conversion of uric acid
to allantoin (more water soluble and easily excreted ).

Fig. Allopurinol inhibits, xanthine oxidase; an example
of competitive inhibition

Lesch-Nyhan Syndrome
 It is an X-linked inherited disorder of purine metabolism.
 Different types of mutations in HGPRTase gene have been
identified in patients with Lesch Nyhan syndrome.
 Incidence is 1:10,000 males.
 There is deficiency ofHGPRTase.
 So, the rate of salvage pathway is decreased resulting in
accumulation of PRPP and decreased level of inhibitory
purine nucleotides.
 The disease is characterized by self mutilation, mental
retardation, excessive uric acid and nephrolithiasis.
 Gout develops in later life.
 The neurological manifestations suggest that the brain is
dependent on the salvage pathway for the requirements of
IMP and GMP.
 Allopurinol is used in treatment, but since salvage pathway is
deficient, it is effective only to inhibit xanthine oxidase.

Hypouricemia -Adenosine Deaminase (ADA) Deficiency
 Both T and B cells are deficient,AR.
 Accumulation of adenosine and dATP; this would inhibit further
production of precursors for DNA synthesis especially dCTP.
 Lymphocytes usually contain high levels of ADA.
 Therefore, ADA deficiency is mainly manifested as reduced
lymphocytes.
 This leads to impaired cellular and humoral immunity.
 Hypouricemia is due to defective breakdown of purine nucleotides.
 Antibiotics and periodic injections of immunoglobulin will be life-
saving.
 Weekly intramuscular injections of bovine ADA were found to be
beneficial.
 Bone marrow stem cells will increase both T and B cells in the
patients.

Hypouricemia -Adenosine Deaminase (ADA)
Deficiency
 Purine nucleotide phosphorylase deficiency also produces
immune deficiency, but only B cell function is affected.
 ADA is sometimes used for the rapid diagnosis of
tuberculosis.
 ADA estimation in CSF is used for the diagnosis of tuberculous
meningitis.
 ADA levels can be estimated in various body fluids like blood,
CSF, pleural fluid, pericardial fluid, ascitic fluid, etc. The usual
cut-off value
 For CSF is 10.0 U/L and for other fluids is 60.0 U/L.
 It has very good sensitivity and specificity for the diagnosis of
pulmonary as well as extrapulmonary tuberculosis.
 Adenosine deaminase (ADA) level in pleural or ascites fluid
may be used for diagnosis for extra-pulmonary tuberculosis.
 A negative test cannot rule out a diagnosis of TB.

Xanthine Oxidase Deficiency
It is a genetic defect.
Characteristic features are hypouricemia, increased
excretion of hypoxanthine and xanthine and liver
damage.

DE NOVO SYNTHESIS OF PYRIMIDINE

The pyrimidine ring (unlike the purine) is
synthesized as free pyrimidine and then it is
incorporated into the nucleotide.

Step 1: Carbamoyl Phosphate Synthesis
 The reaction occurs in cytoplasm (in urea synthesis, the reaction is in
mitochondria).
 The nitrogen of glutamine and bicarbonate react to form carbamoyl
phosphate (step 1).
 The enzyme is carbamoyl phosphate synthetase II (CPS II).
Step 2: Rate Limiting Step
Carbamoyl phosphate and aspartate combine to form carbamoyl
aspartate (step 2).
 The enzyme is aspartyl trans carbamoylase (ATC), which is allosterically
regulated.
 The atoms C2 and N3 are derived from carbamoyl phosphate and the rest
are from aspartate.
Step 3: Formation of Pyrimidine Ring
The 3rd nitrogen and 4th carbon are joined by a covalent bond and
carbamoyl aspartate is cyclized.
 Dihydo orotic acid is produced.
 The enzyme is dihydro orotase (DHOase) (step 3).

Step 4: Oxidation
 Hydrogen atoms are removed from C5 and C6 positions, so that orotic
acid is produced (step 4).
 Enzyme is dihydro orotate dehydrogenase (DHODH).
 It requires NAD as co-enzyme.
Step 5: Formation of OMP
 Ribose-5-phosphate is added to orotic acid, so as to produce orotidylic acid
or orotidine monophosphate (OMP).
 PRPP is the donor of ribose-5-P.
 The enzyme is orotate phosphoribosyl transferase (OPRTase) (step 5).
Step 6: Decarboxylation
 The C7 of OMP is removed as carbon dioxide, so that uridine
monophosphate (UMP) is produced (step 6).
 This is the first pyrimidine that is synthesized.
 The enzyme is OMP-decarboxylase (OMPDC).
 6-aza-uridine inhibits this step, and so used as an anticancer drug.

Step 7: Synthesis of Triphosphates
 UMP is phosphorylated to form UDP (uridine
diphosphate) with the help of ATP (step 7).
 The enzyme is nucleoside monophosphatekinase (UMP
kinase).
 The UDP is phosphorylated to UTP (uridine
triphosphate) with the help of ATP (step 8).
 The enzyme is nucleoside diphosphate kinase.
Step 8: Formation of CTP
 UTP is converted to CTP by adding an amino group
from glutamine catalyzed by CTP synthetase.
 It needs ATP (step 9).

Regulation of Pyrimidine Synthesisi. In eukaryotes the first 3 enzymes, viz, CPS II, ATC and DHOase are present as
a multi-enzyme complex and referred to as ‘CAD', taking the first letters of
the
3 enzymes.
ii. The last 2 enzymes, OPRTase and OMP decarboxylase
are also present as a single functional complex.
Because of this clustering of enzymes, the synthesis is
well coordinated. Both complexes are cytosolic.
iii. The remaining enzyme, dihydro orotate dehydrogenase
(step 4) is mitochondrial.
iv. The major regulatory step in prokaryotes is the
reaction catalyzed by aspartate transcarbamoylase
(ATC) which is allosterically inhibited by CTP.
v. In mammalian cells the regulation occurs at the level
of CPS II (enzyme 1) which is inhibited by UTP
are activated by PRPP. Aspartate transcarbamoylase
(enzyme 2) is inhibited by UTP, and activated by
PRPP.

DE NOVO SYNTHESIS OF P
The pyrimidine ring (unlike the purine) is
synthesized as free pyrimidine and then it is
incorporated into the nucleotide.

Regulation of Pyrimidine Synthesis
i. In eukaryotes the first 3 enzymes, viz, CPS II, ATC
and DHOase are present as a multi-enzyme complex
and referred to as ‘CAD', taking the first letters of the
3 enzymes.
ii. The last 2 enzymes, OPRTase and OMP
decarboxylase
are also present as a single functional complex.
Because of this clustering of enzymes, the synthesis is
well coordinated. Both complexes are cytosolic.
iii. The remaining enzyme, dihydro orotate
dehydrogenase(step 4) is mitochondrial.

Regulation of Pyrimidine Synthesis
vi. Further, OMP decarboxylase is inhibited by UMP.
The requirement of ATP for CTP formation and the
stimulatory effect of GTP on CTP synthetase ensure
a balanced synthesis of purine and pyrimidine
nucleotides.
vii. Pyrimidines can also be salvaged like the purines,
using PRPP and phosphoribosyl transferase and
nucleoside phosphorylase.
viii. Both gene expression and enzyme activity are
regulated.
The first 3 and last 2 enzymes are regulated
by repression/depression.

Disorders of Pyrimidine Metabolism Orotic Aciduria
i. The condition results from absence of either or both of the
enzymes, OPRT ase and OMP decarboxylase.
 It is an autosomal recessive disease.
ii. There is retarded growth and megaloblastic anemia.
 The rapidly growing cells are more affected and hence the anemia.
 Crystals are excreted in urine which may cause urinary tract
obstruction.
 Due to lack of feedback inhibition orotic acid production is
excessive.
iii. The condition can be successfully treated by feeding
cytidine or uridine.
 They may be converted to UTP which can act as feedback
inhibitor.
iv. Orotic aciduria may also occur in ornithine transcarbamoylase
deficiency (urea cycle enzyme) as carbamoyl phosphate
accumulates due to defective conversion to citrulline.

Disorders of Pyrimidine Metabolism Orotic Aciduria
v. Allopurinol competes with orotic acid for the enzyme
orotate phosphoribosyl transferase (enzyme no. 5 ),
leading to orotic aciduria and orotidinuria.
In metabolic disorders related with nucleotides, it is
observed that major clinical consequences result from
overproduction and accumulation of nucleotide
derivatives in cells.
In many cases salvaging and incorporation of abnormal
nucleotide derivatives in DNA during replication can
lead to clinical sequelae often incompatible with life.
Examples are SCID, MNGIE (mitochondrial neurogastro
intestinal encephalopathy syndrome), Lesch Nyhan
syndrome, etc.

Deoxyribonucleotide Formation
Deoxyribonucleotides (both purines and pyrimidine
series) are formed by the reduction at the 2' carbon
of the corresponding nucleoside diphosphates (NDP
to dNDP).
The enzyme is ribonucleotide reductase complex,
which contains non-heme iron.
 It requires NADPH, Thioredoxin and thioredoxin
reductase .
 Thus, UDP is first converted to dUDP, and then to
dUTP.

Combined Regulation of Purine and Pyrimidine Synthesis
Purine and pyrimidines are synthesized in
equimolecular quantities.
This suggests coordinated control of their biosynthesis.
PRPP is the precursor of both purines and pyrimidines.
 The PRPP synthase is inhibited by both purine and
pyrimidine nucleotides.
 Both series of reactions are closely regulated by
allosteric effectors.
 Binding of a specific NTP to the substrate-specificity
site will have positive effect on the reduction of other
NTPs .
Since ATP is required for the reduction of both CDP and
UDP, the purine to pyrimidine balance is always
maintained.

Synthesis of Deoxythymine Nucleotides
The thymine nucleotide is formed by thymidylate
synthase by methylation of dUMP.
 The methyl group is donated by N5,N10-
methylene- THFA.
Later, THFA is regenerated by dihydrofolate
reductase, using NADPH as the reductant .
Methotrexate inhibits the enzyme DHF-reductase.
So dTMP synthesis is inhibited, in turn DNA
synthesis is inhibited.

Anticancer Agents Acting on Pyrimidines
Methotrexate inhibits dihydrofolate reductase and
thereby reduces the regeneration of THFA; it is a
powerful anticancer agent .
5-fluoro-uracil, 5-iodo uracil, 3-deoxy uridine, 6-aza
uridine, 6-aza cytidine and 5-iodo-2-deoxyuridine are
anticancer drugs, which competitively inhibit
thymidylate synthase.
Cytosine arabinoside where ribose is replaced by
arabinose is another anticancer agent.
Two antimetabolites used in treatment of viral
infections, acyclovir (acycloguanosine) and zidovudine
(azido deoxythymidine) are activated by
phosphorylation within cells infected with virus.

Degradation of Pyrimidine Nucleotides
Uracil and Thymine are degraded by analogous
reactions.
 The phosphate is removed from nucleotide to form
corresponding nucleoside.
In the next step, free base is released.
The ring is opened.
Finally, betaamino isobutyric acid is excreted in
urine.
This is the end product of pyrimidines.
Other products are carbon dioxide and ammonia
 Pseudouridine is not metabolized further, and is
excreted as such in urine.

Fig. Catabolism of pyrimidines. Hepatic β-ureidopropionase catalyzes
the formation of both β-alanine and β-aminoisobutryrate from their
pyrimidine precursors.

QUICK LOOK OF CHAPTER
1. A nucleotide is composed of a nitrogenous base, a pentose sugar and phosphate groups esterified to the
sugar.
2. Purine bases in both DNA and RNA are the same.
3. Purine salvage pathway is mediated by two enzymes: Adenine phosphoribosyl transferase (APRTase) and
Hypoxanthine guanine phosphoribosyl transferase (HGPRTase).
4. Committed step of de novo purine synthesis is the reaction catalyzed by amidotransferase.
5. Xanthine oxidase is a metalloflavoprotein containing FAD, molybdenum and iron. Allopurinol inhibits it,
which is a good example of ‘suicide inhibition’.
6. End product of purine catabolism is uric acid. Its normal serum level is 3–7 mg/dL. Level is increased
in gout.
7. Uric acid crystals deposited in the cooler areas of the body cause tophi.
8. Lesh-Nyhan syndrome is a X-linked inherited disorder of purine metabolism, due to deficiency of
HGPRTase. The condition is characterized by selfmutilation tendency.
9. Adenosine deaminase deficiency has been treated by gene replacement therapy.
10. In mammalian cells, regulation of pyrimidine synthesis occurs at the level of Carbamoyl phosphate
synthase II, which is inhibited by UTP.
11. Deficiency of OMP decarboxylase and OPRTase leads to Orotic aciduria.

Nucleotides chemistry and metabolism

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Nucleotides chemistry and metabolism