Download MBBS (Bachelor of Medicine and Bachelor of Surgery) Latest Nucletde Metablsm Lecture PPT
PURINE METABOLISM
? The process of synthesis of complex end
product(s) in a metabolic pathway from simple
precursors molecule is called as de novo synthesis
(de novo = `anew', i.e. starting `from scratch')
? The three processes that contribute to purine
nucleotide biosynthesis are, in order of decreasing
importance.
1.Synthesis from amphibolic intermediates
(synthesis de novo).
2. Phosphoribosylation of purines.
3. Phosphorylation of purine nucleosides.
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 nucleosidases
forming free bases and pentose
sugars.
? Dietary purine bases are
not used for synthesis of tissue
nucleic acids.
? Instead they are degraded to uric acid in the enterocytes.
? Most of the uric acid enters the blood and is eventually
excreted in the urine.
? Humans synthesize the nucleic acids and their derivatives
ATP, NAD+, coenzyme A, etc, from amphibolic
intermediates.
? However, injected purine or pyrimidine analogs,
including potential anticancer drugs, may nevertheless be
incorporated into DNA.
? The incorporation of injected [3H]thymidine into newly
synthesized DNA thus can be used to measure the rate of
DNA synthesis
Denovo
synthesis
Synthesis of purine
base step by step on
the ribose 5phosphate
Synthesis of
purine
nucleotides
Addition of ribose 5-
Salvage
phosphate to the
pathway
preformed purine
bases or addition of
phosphate to the
purine nucleosides
? Denovo purine biosynthesis occurs from basic
precursors and a new purine ring is synthesized
using various metabolic intermediates as
sources of carbon, nitrogen etc.
? This is then used to produce nucleosides and
nucleotides
? In de novo purine
biosynthesis pathway - D-ribose 5
-phosphate is used to synthesize a nucleotide inosine
monophosphate ( IMP).
? This IMP is then converted into AMP and GMP
which are the end products of this pathway.
? There is regulation both at the level of synthesis of
IMP and then its conversion into AMP and GMP.
? Denovo purine biosynthesis is an expensive process
for the cell and uses many metabolic intermediate in
the synthesis of purine ring.
Sources of different atoms of purine ring
N5N10 ?
Methenyl
Tetrahydro
N10 ?Formyl
folate
tetrahydrofolate
Sources of nitrogen and carbon atoms of the purine ring
Denovo synthesis of purine nucleotides
? Purines are synthesized by most of the tissues
? The major site is--- liver .
? Erythrocytes, polymorphonuclear leukocytes &
brain cannot produce purines.
? Subcellular site--- cytoplasm
.
Denovo synthesis of purine nucleotides
SYNTHESIS OF IMP
STEP 1
? Ribose
5-phosphate,
produced in the hexose
monophosphate shunt of
carbohydrate metabolism
is the starting material for
purine
nucleotide
synthesis.
? It reacts with ATP to
form
phsophoribosyl
pyrophosphate (PRPP).
? PRPP
Synthetase
is
inhibited by PRPP
STEP 2
Rate limiting step
? Glutamine transfers it's amide
nitrogen to PRPP to replace
pyrophosphate and produce
5phosphoribosylamine
? The enzyme PRPP glutamyl
amidotransferase is controlled
by feedback inhibition of
nucleoltides (IMP, AMP and
GMP,).
? This reaction is the 'committed.
STEP 3
? Phosphoribosylamine reacts
with glycine in the presence of
ATP to form glycinamide
ribosyl-5-phosphate
or
glycinamide ribotide (GAR)
STEP 4
? N5,N10-formyl-tetrahydrofolate
donates the formyl group and
the
product
formed
is
formylglycinamide
ribosyl
5phosphate.
N5,N10-formyl-tetrahydrofolate
Formylglycinamide ribosyl 5phosphate
STEP 5
? Glutamine transfers the
second amido amino group
Formylglycinamide ribosyl-5-phosphate
to
produce
formylglycinamidine
ribosyl 5phosphate
Formylglycinamidine ribosyl-5-phosphate
STEP 6
? The imidazole ring of
the purine is closed in
an ATP dependent Formylglycinamidine ribosyl-5phosphate
reaction to yield 5-
RING CLOSURE
aminoimidazole ribosyl
5-phosphate
Aminoimidazole ribosyl-5-phosphate
STEP 7
? Incorporation of CO2
(carboxylation) occurs to
yield
aminoimidazole Aminoimidazole ribosyl-5-phosphate
carboxylate ribosyl 5-
phosphate.
? This
carboxylation
reaction does not require
the vitamin biotin and /or
ATP which is the case
with
most
of
the
carboxylation reaction.
Aminoimidazole Carboxylate
ribosyl-5-phosphate
STEP 8
? Aspartate condenses with
aminoimidazole
Aminoimidazole Carboxylate
carboxylate ribosyl 5-
ribosyl-5-phosphate
phosphate.
to
form
aminoimidazole
4-
succinyl
carboxamide
ribosyl 5phosphate
Aminoimidazole -succinyl
carboxamide ribosyl -5 - phosphate
STEP 9
? Adenylosuccinase
or
Adenylosuccinate lyase
cleaves off fumarate and Aminoimidazole -succinyl
carboxamide ribosyl -5 - phosphate
only the amino group of
aspartate is retained to
yield
aminoimidazole
carboxamide ribosyl 5-
phosphate.
Aminoimidazole Carboxamide
ribosyl-5-phosphate
STEP 10
? N10-formyl-THF donates
a one-carbon moiety to
produce
Aminoimidazole Carboxamide
formimidoimidazole
4-
ribosyl-5-phosphate
carboxamide ribosyl 5-
phosphate.
?With this reaction, all the
carbon and nitrogen atoms
of
purine
ring
are
contributed
by
the
respective sources.
Formimidoimidazole Carboxamide
ribosyl-5-phosphate
STEP 11
H2O
Ring closure
IMP cyclohydrolase
Formimidoimidazole Carboxamide
ribosyl-5-phosphate
Inosine monophosphate
(IMP)
The final reaction catalysed by cyclohydrolase leads to ring
closure with an elimination of water molecule from
formimidoimidazole ribosyl-5-P by Inosine monophosphate
(IMP) cyclohydrolase forms IMP.
Synthesis of AMP and GMP from IMP
? Inosine monophosphate is the immediate precursor for the
formation of AMP & GMP
? Aspartate condences with IMP in the presence of GTP to
produce Adenylosuccinate 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. This requires ATP.
STEP-13
STEP-12
STEP-14
Conversion Of IMP to AMP and GMP
STEP-15
MUST REMEMBER
ANTIFOLATE
DRUGS
&
GLUTAMINE
ANALOGS BLOCK PURINE NUCLEOTIDE
BIOSYNTHESIS
Compounds
that
inhibit
formation
of
tetrahydrofolates and therefore block purine
synthesis have been used in cancer chemotherapy.
Inhibitory compounds and the reactions they
inhibit include
? Azaserine -----------------reaction
? Diazanorleucine ---------reaction
? 6-mercaptopurine -------reactions and
? mycophenolic acid -------reaction
FORMATION OF DIPHOSPHATE AND
TRIPHOSPHATE NUCLEOTIDES
? AMP and GMP are phosphorylated using ATP
as the source of phosphate to first make
nucleoside di phosphate and then nucleoside
triphosphate i.e. ADP, ATP, GDP and GTP
CONVERSION OF RIBONUCLEOTIDES TO
DEOXY RIBONUCLEOTIDES
? The synthesis of purine & pyrimidine deoxy ribonucleotides
occur from ribonucleotides by a reduction at the C2 of
ribose moity.
? This reaction is catalyzed by enzyme RIBONUCLEOTIDE
REDUCTASE.
? The enzyme ribonucleotide reductase itself provides the
hydrogen atoms needed for reduction from its sulfhydryl
groups.
? The reducing equivalents, in turn, are supplied by
Thioredoxin, a monomeric protein with two cysteine
residues.
? NADPH-dependent thioredoxin reductase converts the
oxidised thioredoxin to reduced form
? Deoxy ribonucleotides are formed from reduction of
ribo-nucleoside diphosphates.
? Monophosphate and triphosphate are not reduced to
corresponding deoxy ribonucleotides
PURINE SALVAGE PATHWAY
? The free purines ( adenine, guanine & hypoxanthine ) are
formed in the normal turnover of nucleic acids & also
obtained from the dietary sources.
? The free purines are converted to corresponding
nucleotides, & this process is known as `salvage pathway'.
? Adenine phosphoribosyl transferase catalyses the
formation of AMP from adenine.
? Hypoxanthine-guanine
phosphoribosyl
transferase
(HGPRT) converts guanine & hypoxanthine respectively,
to GMP & IMP.
? Phosphoribosyl pyrophosphate (PRPP) is the donor of
ribose 5 phosphate in the salvage pathway.
? The salvage pathway is particularly important in certain
tissues such as erythrocytes & brain where denovo
synthesis of purine nucleotides is not operative.
1. Pu + PR-PP Pu-RP + PPi
? A second salvage mechanism involves phosphoryl
transfer from ATP to a purine ribonucleoside (Pu-R):
Pu-R + ATP PuR-P + ADP
? Phosphorylation of the purine nucleotides, catalyzed
by adenosine kinase converts adenosine and
deoxyadenosine to AMP and dAMP.
Or
REGULATION OF PURINE NUCLEOTIDE
BIOSYNTHESIS
? The de novo biosynthesis is regulated in two
ways
1. Regulation of IMP formation
2. Regulation of AMP and GMP formation from
IMP.
1. Regulation of IMP formation
? Occurs at first two reaction catalyzed by
i. PRPP synthase
ii. Glutamine phosphoribosyl ? amido-
transferase.
Though both are regulatory enzymes, the
second step catalyzed by amidotransferase is
also a committed step for purine synthesis.
Hence it is more important.
i. REGULATION OF PRPP SYNTHASE
? The overall determinant of the rate of de novo purine
nucleotide biosynthesis is the concentration of PRPP.
? This, in turn, depends on the rate of PRPP synthesis,
utilization, degradation, and regulation.
? The rate of PRPP synthesis depends on the availability
of ribose 5-phosphate and on the activity of PRPP
synthase
? Activity of PRPP synthase is allosterically inhibited by
both the adenosine and guanosine nucleotides i.e
AMP, ADP, GMP and GDP.
ii. REGULATION OF AMIDOTRANSFERASE
? Amidotransferse is feedback inhibited by products i.e.
AMP, ADP, GMP and GDP.
? AMP and GMP act as competitive inhibitors.
? So, at a high PRPP (substrate ) concentration, AMP and
GMP will not be able to inhibit the amidotransferase
enzyme.
? Amidotransferse is stimulated by its substrate PRPP.
(feed forward reaction)
? A very high PRPP concentration will lead to increased
purines and their catabolism producing hyperuricemia.
Regulation of AMP and GMP formation from IMP
? AMP feedback inhibits its own synthesis at the
adenylosuccinate synthase level.
? Simultaneously ATP stimulate GMP synthesis at
xanthine transaminidase step.( cross regulation)
? Similarly GMP inhibits its own synthesis at the IMP
dehydrogenase step.
? GTP
stimulates
AMP
synthesis
at
the
adenylosuccinate synthase step. ( cross regulation)
? This cross regulation ensures that adenine and
guanine nucleotide synthesis is in equal proportion. If
AMP is decreased, it stimulates its own synthesis and
inhibits GMP synthesis and vice versa.
REGULATION OF PURINE
NUCLEOTIDE BIOSYNTHESIS
? Regulation of IMP is
shown by solid lines
? AMP and GMP synthesis
are shown by dotted lines
E1 = PRPP Synthetase
E2 = Amido Transferase
E3 = Adenylosuccinate
synthase
E4 = IMP dehydrogenase
E5
E5 = Xanthine
transaminidase
DEGREDATION OF PURINE NUCLEOTIDES
1. The end product of purine metabolism in humans is
uric acid.
2. The nucleotide monophosphates (AMP , IMP &
GMP) are converted to their respective nucleoside
forms (adenosine,inosine & guanosine ) by the
action of nucleotidase.
3. The amino group, either from AMP or adenosine,
can be removed to produce IMP or inosine
respectively by deaminase.
4. Inosine & guanosine are, raspectively , converted to
hypoxanthine & guanine (purine bases) by purine
nucleoside phosphorylase.
.
5. Adenosine is not
degreded by this enzyme, hence it has
to be converted to inosine by deaminases.
6. Guanine undergoes deamination by guanase to form
xanthine.
7. Xanthine oxidase is an important enzyme that converts
hypoxanthine to xanthine, & xanthine to uric acid.
8. This enzyme contains FAD, Molybdenum & Iron, & is
mainly found in liver & small intestine.
9. Uric acid ( 2,6,8-trioxopurine ) is the final excretory
product of purine metabolism in humans.
10. Uric acid can serve as an important antioxidant by
getting itself converted non enzymatically to allantoin.
Guanase
DISORDERS OF PURINE METABOLISM
1. Hyperuricemia And Gout
2. Lesch-Nyhan syndrome
3. Severe Combined Immuno Deficiency (SCID)
4. Purine Nucleoside Phophorylase Deficiency
URIC ACID
1. Uric acid (2,6,8-trioxopurine) is the end product of
purine metabolism in humans.
2. The normal concentration of uric acid in the serum of
adults is in the range of 3-7 mg / dl.
3. In women, it is slightly lower (by about 1mg) than in
men.
4. The daily excreation of uric acid is about 400-600 mg.
It is filtered , reabsorbed and secreted by kidney tubules.
5. At the pH of 5.75 and
above, it forms monosodium
urate salt which is 10 times more soluble than uric
acid.
6. In plasma (pH of 7.4), monosodium urate is
predominant form present which is relatively more
soluble.
7. Any condition which decreases blood pH (acidosis)
,therefore, promotes the formation of insoluble uric
acid than the more soluble monosodium urate.
1. HYPERURICEMIA AND GOUT
? Increase in blood uric acid level above the normal
value of >7 mg% is called hyperuricemia.
? This is sometimes associated with increased uric acid
excreation ( Uricosuria)
? In severe hyperuricemia, crystals of sodium urate get
deposited in the soft tissues, particularly in the joints.
? Such deposits are commonly known as TOPHI.
? This causes inflammation in the joints resulting in a
painful arthritis.
? Sodium urate &/or uric acid may also precipitate in
kidneys & ureters that result in renal damage & stone
formation.
GOUT
? Hyperuricemia leading to arthritis is called gout.
? Gout is often called a disease of bones and stones due
to recurrent stones formation and inflammmation of
joints.
? Gouty arthritis is debilitating painful condition
leading to deformity of joints.
? Typical
gouty
arthritis
affects
first
metatarsophalangeal joint.(GREAT TOE)
TYPES OF GOUT
Two types:
1. Primary ?
due to metabolic defect of uric acid where its
synthesis as such is increased.
2. Secondary ?
- Due to increased nucleotide turn over
wherein more uric acid is formed .
- Uric acid metabolism is normal.
- This occurs as a consequence of some
other primary disease associated with
increased tissue catabolism
CAUSES OF GOUT
? PRIMARY GOUT
1. PRPP synthetase over activity due to defective enzyme
varient forms of PRPP synthetase-which are not
subjected to feedback regulation-have been detected.
This leades to increased production of purines.
2. PRPP-glutamyl-amidotransferase - defective enzyme
The lack of feedback control of this enzyme by purine
nucleotides also leads to their elevated synthesis.
3.HGPRTase deficiency or Lesch-Nyhan syndrome
? Deficiency of
HGPRTase causes a decrease in salvage
pathway of hypoxanthine and guanine to reform
nucleotide.
? This in turn spares PRPP and results in overproduction
of purine nucleotides and their degradation to uric acid.
4. Glu-6-phosphatase dificiency or Von-Gierke's
disease
? When this enzyme is deficient, glucose-6-phosphate
cannot be converted to glucose.
? So more glucose-6-phosphate is channeled into the
pentose phosphate shunt pathway, resulting in
increased availability of ribose-5-phosphate.
? This would lead to increased formation of PRPP
ultimately, purine over production.
? 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
SECONDARY GOUT
A. Increased Production of Uric Acid
i. Rapidly growing malignant tissues, e.g.
leukemias, lymphomas, polycythemia.
ii. Cancer patients on radiotherapy or
chemotherapy (tumor lysis syndrome) due
to increased cellular turnover
iii. Increased tissue damage due to trauma and
raised rate of catabolism as in starvation
iv Psoriasis ? skin disease
Secondary Hyperuricemia
B. 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
? 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
? 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.
TREATMENT
? Reduce dietary purine intake and restrict alcohol.
? Increase renal excretion of urate by uricosuric drugs,
which decrease the reabsorption of uric acid from kidney
tubules, e.g. probenecid.
? Reduce urate production by allopurinol, an analog of
hypoxanthine and 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`).
? Colchicine, an anti-inflammatory agent is very useful to
arrest the arthritis in gout.
Lesch-Nyhan Syndrome
? Inability of the body to salvage hypoxanthine and
guanine due to the complete deficiency of HGPRTase
(Hypoxanthine-Guanine phosphoribosyl transferase)
? It is an X-linked inherited disorder of purine
metabolism, the disease is limited to males only
? Different types of mutations in HGPRTase gene have
been identified in patients with Lesch Nyhan
syndrome.
? Incidence is 1:10,000 males.
? HGPRT deficiency results in the accumulation of
PRPP and decrease in GMP and IMP.
? Increased level of Hypoxanthine and Guanine
in degradation to uric acid
? Also PRPP accumulates
stimulates production of Purine nucleotides
increases their degradation to uric acid
? Leads to hyperuricemia---Gout-like symptoms
Nephrolithiasis ( Renal stones)
Neurological symptoms
? self mutilation
? spasticity,
? aggressiveness,
? mental retardation
DIAGNOSIS
? Increase urinary urate / creatinine ratio
? Absent / reduced enzyme activity in
lymphocytes or fibroblast
? Mutation analysis of Hypoxanthine-Guanine
phosphoribosyl transferase (HGPRT) gene.
Severe combined immunodeficiency (SCID)
? The deficiency of adenosine deaminase (ADA) causes
severe
combined
immunodeficiency
(SCID)
involving T-cell and usually B-cell dysfunction.
? ADA deficiency results in the accumulation of dATP.
? dATP is an inhibitor of ribonucleotide reductase
which causes reduced synthesis of other dNTPs and
therefore DNA synthesis and cell replication is
inhibited.
? Thus proliferation and differentiation of immune cells
is compromised.
SCID
? 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.
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.
SCID - Treatment
? Antibiotics and periodic injections of
immunoglobulin will be lifesaving.
? Bone marrow stem cells will increase both T
and B cells in the patients.
? Enzyme replacement therapy with ADA-
Polyethylene glycol ( the first successful
application of enzyme replacement therapy for
an inherited disease.
? Gene therapy- recently, ADA gene has been
successfully transfected into stem cells of
ADA deficient children.
Purine Nucleoside Phophorylase
Deficiency
? Less severe than ADA deficiency
? Associated with severe deficiency of T- cells
but apparently normal B- cell function.
? Immune dysfunction appear to result from
accumulation of dGTP, and dATP, which
inhibit ribonucleotide reductase and thereby
deplete cells of DNA precursors.
This post was last modified on 30 November 2021