Outline

 Nucleotide Metabolism:
o Purine Metabolism
• de novo salvage pathways
• salvage pathways
o Pyrimidine metabolism
• Inborn errors of Nucleotide Metabolism
Recaps of purines & pyrimidines
 METABOLISM OF NUCLEOTIDES

Mammals and most of the lower

vertebrates can synthesise purines
pyrimidines and are said to be
phototrophic
 There are two pathways leading to nucleotides

 De novo synthesis: The synthesis of nucleotides begins with their

metabolic precursors: amino acids, ribose-5-phosphate, CO2, and
one-carbon units.
 Salvage pathways: The synthesis of nucleotide by recycle the free
bases or nucleosides released from nucleic acid breakdown.

 The significant of salvage pathway

 1- Save fuel
 2- Some tissues and organs such as brain and bone marrow are
only capable of synthesizing nucleotides by Salvage pathways
– Broken down endogenous nucleotides = salvage pathways
Biosynthesis of Purine Nucleotides
 Purine nucleotides are involved in many cellular functions
 components of DNA and RNA,
 sources of energy,
 enzyme cofactors,
 components of signal transduction.
 The three processes that contribute to purine nucleotide
biosynthesis are.
 Synthesis from amphibolic intermediates ( synthesis de novo ).
 Phosphoribosylation of purines.
 Phosphorylation of purine nucleosides.
 Purines are synthesized by most of the tissues ,
 The major organ location liver.
 Subcellular location cytoplasm
 Biosynthesis of Purine Nucleotides

1.Denovo synthesis(Major pathway)

 Synthesis of purine nucleotides from various small molecules
derived as intermediates of many metabolic pathways in the
body.

2.Salvage pathway: Minor pathway

 De Novo Synthesis

 The atoms of the purine ring originate from a wide variety of

sources
 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.
 purine nucleotide
 purine nucliotide first synthesised is INOSINE MONO
PHOSPHATE (IMP)
 It is a nucleotide composed of (HYPOXANTHINE + RIBOSE
+ PHOSPHATE )
 From IMP other purine nucleotides are synthesized, like
 AMP(adenosine mono phosphate)
 GMP( guanosine mono phosphate)
Steps of De Novo Synthesis of Purine
 Step 0 (Preparatory Step), PRPP synthesis
 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)

Structure of Phosphoribosyl pyrophosphate (PRPP)

 Steps of De Novo Synthesis of Purine (1-5)

 The de novo synthesis of one molecule of

purine nucleotide requires 6 ATP

1 =Phosphoribosyl
amidotransferase=
rate limiting.
2 = GAR synthetase
3 = GAR
transformylase
4 = FGAR amido
transferase=inhibite
d by azaserine
5 = Cyclase
Steps of De Novo Synthesis of Purine (6-8)

6 =AIR-carboxylase
7 = SAICAR synthetase
8 = SAICAR lyase
Steps of De Novo Synthesis of Purine (9-11)

9 = AICAR transformylase
10 = IMP synthase
11 = Adenylosuccinate
synthetase & adenylosuccinase
Synthesis of inosinic acid
 Synthesis of AMP and GMP from IMP

 Inosine monophosphate is the immediate precursor for

the formation of AMP & GMP
 Aspertate condences with IMP in the presence of GTP to
produce sdenylosuccinate which on cleavage forms AMP.
 For the synthesis of GMP, IMP undergoes
NAD+ dependent dehydrogenation to form Xanthosine
monophosphate ( XMP). Glutamine then transfers amide
nitrogen to XMP to produce GMP.
Synthesis of AMP and GMP from IMP
 Purine Salvage Pathways
 This pathway ensures the recycling of purines formed by degradation
of nucleotides.
 Nucleosides and deoxy-nucleosides can also be salvaged.
 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.
 The free purines are salvaged by two different enzymes;
 adenine phosphoribosyl transferase (APRTase) and
 hypoxanthine guanine phosphoribosyl transferase
(HGPRTase).
 The pathway is of special importance in tissues like RBCs and brain
where the de novo pathway s not operating.
 The salvage pathway economize intracellular energy expenditure.
 Regulation of Purine Nucleotide Biosynthesis

 The purine nucleotide synthesis is well coordinated to

meet the cellular demands.
 The intracellular concentration of PRPP regulates
purine synthesis to a large extent.
 This inturn is dependent on the availability of ribose-
5 -phosphate & the enzyme PRPP synthetase
 PRPP glutamyl amidotransferse is controlled by a
feedback mechanism by purine nucleotides.
 Regulation of Purine Nucleotide Biosynthesis
 If AMP & GMP are available in adequate amounts to meet
the cellular requirements, their synthesis is turned off at
the amidotransferase reaction.
 Another important stage of regulation is in the convertion
of IMP to AMP & GMP.
 AMP inhibits adenylosuccinate synthetase while GMP
inhibits IMP dehydrogenase.
 Thus, AMP & GMP control their respective synthesis
from IMP by a feedback mechanism.
Degradation of Purine Nucleotides

 The end product of purine nucleotide catabolism in humans is

uric acid (urate)
 This degradation is taking place mainly in the liver
 The nucleotide monophosphates (AMP, IMP & GMP ) are
converted to their respective nucleoside forms (adenosine,inosine
& guanosine ) by the action of nucleosidase.
 The amino group, either from AMP or adenosine, can beremoved
to produce IMP or inosine respectively
 The xanthine oxidase is a metalloflavoprotein containing FAD,
molybdenum and iron.
 As xanthine is oxidized to uric acid, the electrons are transferred
first to molybdenum, then to FAD, and finally to molecular
oxygen,
 when hydrogen peroxide is produced.
 Degradation of Purine Nucleotides

Main pathway is in red arrows. PNP = purine nucleoside

phosphorylase; R-1-P = ribose-1-phosphate
The major pathways of purine catabolism in
animals.
 Inborn errors of Nucleotides metabolism

 Shows  broad:
o neurological
o Immunological
o Haematological & renal manifestations
o  Limited awareness of the phenotypic spectrum, Due to
genetic heterogeneity 
o Orotic aciduria =pyrimidine nucleotide synthesis
o hyperuricemia
o Gout
 Disorders of Purine Metabolism

1. HYPERURICEMIA AND GOUT

 Uric acid is the end product of purine metabolism in
humans
 Normal blood level of uric acid ranges from 2–5mg/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
 Disorders of Purine Metabolism
 Hyperuricemia refers to an elevation in the serum uric acid
concentration.
 Usually serum uric acid concentration exceeding 7 mg/ dL in
male and 6 mg/dL in female.
 This is sometimes associated with increased uric acid
excreation (Uricosuria)
 The manifestations are due to the low solubility of uric acid in
water
 GOUT

 is metabolic disease associated with overproduction of uric

acid.
 At the physiological pH, uric acid is found in a more soluble
form as sodium urate.
 In severe hyperuricemia, crystals of sodium urate get
deposited in the soft tissues, perticularly in the joints.
 GOUT

 Gout is a chronic disorder characterised by

 Excess of uric acid in blood (Hyperuricemia)
 Deposition of sodium monourate in alveolar and nonalveolar
structures producing so called tophi.
 Recurring attacks of acute arthritis.
 These are due to deposition of monosodium urate in and
around the structures of the affected joints
 This causes inflammation in the joints resulting in a painful
gouty arthritis. Typical gouty arthritis affects first
metatarsophalangeal joint.(GREAT TOE)
 GOUT
 Historically, gout was found to be often associated with high
living, over-eating & alcoho consumption.
 The prevalence of gout is about 3 / 1,000 persons, mostly
affecting males.
 Clinical features:

 Attacks are precipitated by alcohol intake.Often patient have

few drinks , go to sleep symptomless , but are awakened
during early hours by severe joint pains
 Synovial fluid shows birefringent crystals under polar
microscope is diagnostic.
 Types of GOUT
 There are two main types of gout:
1. Primary gout,
2. Secondary gout.
3. Pseudogout
 1.PRIMARY GOUT.
 It is an inborn error of metabolism due to overproduction of
uric acid.
 This is mostly related to over production of purine nucleotides.
 PRPP synthetase : in normal circumstances , PRPP synthetase
is under feedback control by purine nucleotides ( ADP &
GDP ).
 However, varient forms of PRPP synthetase-which are not
subjected to feedback regulation-have been detected. This
leades to increased production of purines.
PRPP glutamylamidotransferse :
The lack of feedback control of this enzyme by purine
nucleotides also leads to their elevated synthesis.
HGPRT deficiency : This is an enzyme of purine salvage
pathway, & its defect causes Lesch-Nyhan syndrome.
This disorder is associated with increased synthesis of
purine nucleotides by a two fold mechanism.
Firstly, decreased utilization of purines ( Hypoxanthine &
guanine ) by salvage pathway, resulting in the
accumulation & divertion of PRPP for purine nucleotides.
Secondly, the defect in salvage pathway leads to
decreased levels of IMP & GMP causing impairment in
the tightly controlled feedback regulation of their
production.
 Glucose 6-phosphatase dificiency:
 In type I glycogen storage disease ( von-gierke’s ), glucose-6-
phosphate cannot be converted to glucose due to the deficiency
of glucose-6-phosphatase.
 This leads to the increased utilization of glucose-6-phosphate by
HMP shunt resulting in elevated levels of ribose-5-phosphate &
PRPP &, ultimately, purine overproduction.
 von gierke’s disease is also associated with increased activity of
glycolysis.
 Due to this, lactic acid accumulates in the body which interferes
with the uric acid excretion through renal tubules
 Elevation of Glutathione Reductase :

 varient of glutathione reductase generates more NADP+ which

is utilized by HMP shunt .
 This leads to increased ribose 5-phosphate and PRPP
synthesis.
 2.Secondary gout:

 Secondary hyperuricemia is due to various diseases causing

increased synthesis or decreased excretion of uric acid.
 Increased degradation of nucleic acids (hence more Uric acid
formation) is observed in various cancers (leukemias,
polycythemias, lymphomas, etc).
 Psoriasis and increased tissue breakdown (trauma, starvation
etc).
 3.Pseudogout

 The clinical manifestations of pseudo gout are similar

to gout.
 This disorder is caused by the deposition of calcium
pyrophosphate crystals in joints.
 Further serum uric acid concentration is normal in
pseudo gout.

Location of gout attack

 Treatment of Gout: Consist of:

(a) Palliative Treatment

(b) Specific Treatment
 a) Palliative treatment:

Bed rest in acute stage,

Diet—Purine free diet, Restricting alcohol consumption.
 Anti-inflammatory Drugs
1.Colchicine:
 One of the nonspecific antiinflammatory drug.
 no effect on urate metabolism or excretion.
 Colchicine therapy is instituted during acute attack
Mechanism:
 Suppresses the synthesis and secretion of the chemotactic factor that is
produced in urate crystal-induced inflammation.
Dosage: Available as 0.5 mg tablet
In acute gout: One tablet hourly till symptoms are relieved or diarrhoea occurs.
Long-term management: One tab 3 to 4
times a week.
 2. NSAIDS

 Drugs like:
 Indomethacin,
 Diclofenac,
 Naproxen,
 Piroxicam, Inhibit PGs
 Fenoprofen, synthesis which are
important mediator
 Flurbiprofen, of the inflammatory
 Ibuprofen, response
 Rofecoxabin
 (b) Specific Treatment
 Aim: To lower the uric acid level of blood.
 Methods: The above can be achieved in 3 ways
 1. By increasing the renal excretion of uric acid (uricosuric
drugs).
 2. By decreasing the synthesis of uric acid using enzyme
inhibitor
 3. By increasing oxidation of uric acid.
 1. Uricosuric Drugs
 uricosuric agent which is enhances the renal excretion of uric
acid probably by specific inhibition of its tubular reabsorption
or secretion
 Examples:
 Salicylates (LD 1 - 2 gm/day or HD 5 to 6 gm/day
 Probenecid (Benemide) (Available as 500 mg tablet)
 ½ tablet twice daily
 Halofenate (good uricosuric effect)
 can be safely used for short-term and long-term therapy
 Note: Uricosuric drugs are effective provided renal
function is normal.
 2. Enzyme Inhibitor
O
 Allopurinol O
(zyloprin) drug of choice for the treatment of
primary gout
C C H
N of hypoxanthine
 is a structural analog C
HN C HN C
 Acts by competitive CH inhibition on “xanthine oxidase” and thusN
uric acid synthesis is impaired HC C
HC C
 Further allopurinol
N N is oxidized to alloxanthineNby xanthineN
oxidase. H H
Hypoxanthine
 Alloxanthine, Allopurinol
in turn is a more effective inhibitor of xanthine
oxidase. This type of inhibition is referred to as suicide
inhibition.
 Dosage: Available as 100 mg tablet Initially 100 to 200 mg
daily. Maintenance: 200 to 600 mg daily. Not recommended in
children.
 Note:Also used for secondary hyperuricaemia
inhibitory action on the enzyme tryptophan pyrrolase.
 3. Drugs Increasing Uric Acid Oxidation

 Urate oxidase:
 used for lowering of uric acid level by oxidising uric acid.
Dosage:
 10,000 IU daily for 10 days.
 It shows a significant decrease in uric acid level.
 Can be used in severe gout with renal involvement &
secondary hyperuricaemia.
 Notes:
 Besides the drug therapy, restriction in dietary intake of
purines and alcohol is advised.
 Consumption of plenty of water will also be useful.
Action of medicines in gout
 Lesch-Nyhan Syndrome
 Fist described tn 1964 by Michael Lesch( a medical student)
and William L. Nyhan (his teacher).
 X-linked inherited disorder of purine metabolism
 Only males are affected by this.
 It is X-linked recessive defect of hypoxanthine-guanine
phosphoribosyltrans-ferase (HGPRTase).
 An enzyme of purine salvage pathway
 Incidence is 1:10,000 males
 HGPRT deficiency results in the accumulation of PRPP and
decrease in GMP and IMP, ultimately leading to increased
synthesis and degradation of purines
 Lesch-Nyhan Syndrome is characterized by
 Neurological abnormalities
 mental retardation,
 aggressive behavior,
 learning disability
 self mutilation (fingers , lips)
 excessive uric acid and
 Gout develops in later life
Treatment : Allopurinol
PY
RI M
ID
INE
ME
TA
BO
LI S
M
 SYNTHESIS OF PYRIMIDINES

 Pyrimidine is a monocyclic ring

 The synthesis of pyrimidines is a much simpler
process compared to that of purines.
 aspartate, gutamine and CO2 contribute to atoms in
the formation of pyrimidine ring
 Pyrimidine ring is first synthesized and then attached
to ribose 5-phosphate.
 this is in contrast to purine nucleotide synthesis
where in purine ring is built upon a pre-existing
ribose5-phosphate.
Sources of C and N atoms of pyrimidine
Materials required for synthesis of pyrimidines
 Carbamoyl phosphate: Synthesised from CO2 & Glu
 PRPP: 5-phosphoribosyl-1-pyrophosphate
 Various enzymes:
 Carbamoyl phosphate synthetase II (CPS-II)
 Transcarbamoylase
 Dihydro-orotase
 Dehydrogenase
 Transferase and
 Decarboxylase
 ATP: For energy
 Amino acid: Aspartic acid
 Cofactors: FAD+, NAD+, Mg++
Steps of Synthesis of pyrimidine (1-5)
1. CPS II
2. Aspartate
Transcarbamoylase
(ATC
3. Dihydro-orotase
(DHOase)
4. Dihydro-orotate
Dehydrogenase
(DHODH
5. Orotate Phospho
Ribosyl
Transferase
(OPRTase)
Steps of Synthesis of pyrimidine (6-9)
6.OMP decarboxylase(OMPDC
7.UMP Kinase (UMOK)
8.Nucleoside diphosphate Kinase
9. CTP synthetase
Metabolic pathway for the synthesis of pyrimidine
 Salvage Pathway of Pyrimidine Synthesis
 The pyrimidines (like purines) can also serve as precursors in
the salvage pathway to be converted to the respective
nucleotides.
 This reaction is catalyzed by pyrimidine phospshoribosyl
transferase which utilizes PRPP as the source of ribose 5-
phosphate.
 Significance
 Salvage pathway provide a pathway for the utilization of
pyrimidine bases derived from diet(exogenous) and normal
turnover of nucleic acids.
 Inhibitors of pyrimidine synthesis
 Sulfonamides
 Methotrexate
 Trimethoprim
 5-fluorouracil
 Fluorocytosine
 Regulation of pyrimidine synthesis:

 In eukaryotes the first 3 enzymes, ( CPS II, ATC & DHOase

are present as a multi-enzyme complex ‘CAD', taking the 1st
letters of the 3 enzymes.
 The last 2 enzymes, OPRTase and OMP decarboxylase are
present as a single functional complex. Due to clustering of
these enzymes , the synthesis is well coordinated
 The remaining enzyme, dihydro orotate dehydrogenase
(step 4) is mitochondrial.
 The major regulatory step in prokaryotes is the reaction
catalyzed by aspartate transcarbamoylase (ATC) which is
allosterically inhibited by CTP.
 Regulation of pyrimidine synthesis:

 CPSII is main regulatory enzyme in mammalian cells.

 inhibited by UTP & activated by PRPP
 Aspartate transcarbomylase :
 main regulatory enzyme in prokaryotes.
 inhibited by CTP & activated by ATP
 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
 Catabolism of Pyrimidine
 The pyrimidine nucleotides undergo similar reactions
( dephosphorylation, deamination and cleavage of
glycosidic bond) like that of purine nucleotides to liberate
the nitrogenous bases cytosine, uracil and thymine.
 Uracil and Thymine are degraded by analogous reactions.
 The bases are then degraded to highlyl soluble products β-
alanine and β-aminoisobutyrate.
 Other products are carbon dioxide and ammonia
Disorders of pyrimidine metabolism
 OROTIC ACIDURIA:

 Orotic aciduria type I

 deficiency of Orotatephosphoribosyl transferase and OMP –
decarboxylase.
 Orotic aciduria type II :
 Rare, deficeincy of ONLY OMP decarboxylase.
 Both types are inherited as autosomal recessive disorders.
 Features of Orotic Aciduria
 Due to lack of feedback inhibition orotic acid
 production is excessive.(UMP inhibits OMP decarboxylase)
 Rapidly growing cells are affected – anemia
 Retarded growth
 Crystals excreted in urine causing urinary obstruction.
 Both types respond to uridine , as it is converted to UTP .
 This acts as feed back inhibitor.
 Other causes of orotic aciduria
 Deficeincy of liver mitochondrial ornthine – trancarbomylase
(X-linked).

 under utilized substrate carbomyl phosphate enters cytosol

 Stimulates pyrimidine nucleotide biosynthesis

 Leading to orotic aciduria

 Drugs may precipitate orotic aciduria

 Allopurinol: an alternative substrate for orotate

phosphoribosyl transferase

 competes with natural substrate orotic acid.

 The resulting nucleotide product inhibits OMP

DECARBOXYLASE

 leading to Orotic aciduria and orotiduniria

 Drugs may precipitate orotic aciduria

 6-Azauridine:following its conversion to 6-azauridylate,

 Competively inhibits orotidylate decarboxylase

 Leads to increased excretion of orotic acid (orotic aciduria)

and orotidine (orotidinura)
 Anticancer Agents Acting on Pyrimidines

 Methotrexate
 inhibits dihydrofolate reductase(DHF) and
 Leads reduces the regeneration of tetrahydrofolic acid (THFA)
 dTMP synthesis is inhibited,
 in turn DNA synthesis is inhibited
 it is a powerful anticancer agent
 Anticancer Agents Acting on Pyrimidines
 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.
 Reye’s syndrome:
 This is considered as a secondary orotic aciduria.
 It is believed that a defect in ornithine
trascarbamoylase (or urea cycle ) causes the
accumulation of carbamoyl phosphate.
 This is then diverted for the increased synthesis and
excretion of orotic acid