BL2- Purine Metabolism by prof unikl rcmp.pdf

biochemistry preclinical

1. NucleotideMetabolism
2 lectures by AP DrN.Muniandy
Aims andObjectives:
At the end of the lectures the students are expectedto;
1.Understand how purine and pyrimidine nucleotides are
synthesized through denovo and salvage pathways, and the
involvement of folic acid and vitaminB12
2.Explain the catabolism of purine and pyrimidinenucleotides
3.Understand the regulation of denovo biosynthesis of purines
and deoxyribonucleotide synthesis
4.Appreciate the connection between hyperurisaemia andgout
and the use of anti-folate drugs andanalogues

2. BL2 Purine metabolism and Uric acidformation
DrN.Muniandy(19.3.2019)
LectureOutline:
1.Digestion of dietary nucleicacids
2Roles of Nucleotides in biochemicalprocesses
3.Source of each C and N atom in the purine and pyrimidine molecularskeleton
4.Purinebiosynthesis:
1.Denovo biosynthesis of purinenucleotides
2.Regulation of Denovo biosynthesis of purinenucleotides
3.Salvage pathway for synthesis ofpurines
5.Catabolism of purinenucleotides
6.Uric AcidChemistry
7.Hyperurisaemia andgout.
8.Clinical syndromes ofhyperuricaemia
9.Diagnosis and treatment ofgout.

3. 3
Nucleotidescontain:
bases-purines (Adenine &Guanine)
-pyrimidines (Cytosine, Uracile,Thymine)
-ribose ordeoxyribosesugar
phosphate
Nucleosides

4. NH
2 at 12
0
clock
NH
2 at 7
0
clock
NH
2 at 12
0
cl,C4
CH
3 atC5
9membered
ring
6membered
ring

5. Arrangement of nucleotides within
the structure of nucleicacids

6. NH
2 at 12
0
clock
NH
2 at 7
0
clock
NH
2 at 12
0
cl,C4
CH
3 atC5

7. 1. Digestion of dietary nucleicacids
Dietary purines &
pyrimidines are not
used to a largeextent
for the synthesis of
tissue nucleicacids.
The purines are
generally convertedto
uric acid by intestinal
mucosal cells and
excreted in theurine.
The remainder of
dietary purines are
metabolized bythe
intestinalflora

8. 2.RolesofNucleotides in biochemicalprocesses
1.Nucleotides are the activated precursorsfor synthesis of DNA&
RNA.
2.Adenine nucleotide, ATP, is the universal currency ofenergy.
GTP also serves as an energy source for a select group ofbiological
processes.
3.Nucleotide derivatives participate in biosyntheticprocesses.
Eg.UDP-glucose
CDPdiacylglycerol
-biosynthesis ofglycogen
-biosynthesis ofphosphoglyceride
S-AdenosylMethionone-methyl groupcarrier
4.Adenine nucleotides are essential components ofcoenzymes.
Eg.NAD+,FAD,CoA (adenine, ribose, pantothenic acid,mercaptoethylamine)
5.Nucleotides also play role as metabolic regulators in signal-
transductionpathways.
Eg.i)cAMPand cGMP as 2
nd
messengers in signaltransmission
ii)ATP, ADP, AMP and other nucleotides -ATP acts as the donor of
phosphoryl groups transferred by proteinkinases.

9. 3. Nucleotidebiosynthesisby; 1.denovo
2. salvagepathway
Sources of C and N atoms in purine andpyrimidine
N-1 fromaspartate
C-6 fromCO
2
N-3, N-9 fromglutamine
C-4, C-5 and N-7 are fromglycine
C-2 and C-8 from N10 formyl tetrahydrofolate
N-1, C-4, C-5, C-6 fromaspartate
C-2 fromCO
2
N-3 from Amide N ofglutamine
6
1

10. 10
BasicCompounds
denovo synthesisof
purine &pyrimidine
FreeBases
synthesisthrough
salvagepathway
NMP
Nucleotide pool incell
NDP NTP
Ribonucleotide
reductase
dNDP dNTP
Nucleotides (ribonucleotide and or deoxyribonucleotide)
used in various biochemicalprocesses
(ribonucleotides)
(deoxyribo
nucleotides)

11. 4. Purinebiosynthesis-Introduction
•Requirements for the nucleotides and basesare met by
both dietary intakeor de novosynthesisfrom low MW
precursors.
•The salvage pathways are a major source of nucleotides
for synthesis of DNA, RNA andco-factors.
•Synthesis proceeds by the synthesis of the purine base
upon the ribose sugarmoiety.
•Both the salvageand de novosynthesis pathways of
purine biosynthesis lead an activated sugar intermediate,
5-phosphoribosyl-1-pyrophosphate(PRPP).

12. 4.1Denovo biosynthesis of purinenucleotides
•Requires ribose-5-phosphate, aspartate, glycine,
glutamine, N
10-formyl tetrahydrofolate & CO
2.
•Inosine 5'-monophosphate (IMP) is a common
intermediate for AMP andGMP.
•Starting from IMP, synthesis of AMP and GMP follows
different pathways
•Denovo biosynthesis of purine is energydemanding.
•Erythrocytes, brain, PMN do not carry outDenovo
synthesis

13. PRPP is synthesizedfromribose-5-phosphate(intermediateofPPP)
by the action of PRPP synthetase andATP
ribose-5-phosphate +ATP ---------------->PRPP +AMP
PRPP synthetase

14. 14
4.1.1Summary of denovo synthesis ofpurine
glutamine PRPP
amidotransferase (committedstep)PRPP synthetase
Ribose5 phosphate PRPP
ATP
5-phosphoribosyl-1-amine(PRA)
glutamine
glycine
N
10
-formyltetrahydrofolate
CO
2
aspartate
Inosine5’-monophosphate
(IMP)
guanosineMP
(GMP)
adenosineMP
(AMP)
ATP
GTP
PRA

15. Denovo synthesis of purine
hypoxanthine

16. Pathways from inosine 5’-monophosphate (IMP)
to GMP andAMP.
4.1.2Pathway from IMPto
GMP &AMP:
IMP is a branch point for the synthesis of
GMP orAMP
a)Energy requirements for theAMP
pathway are met byGTP
Adenosylsuccinate synthetaseis
inhibited byAMP.
b)Energy requirements for the GMP
pathway (GMP synthetase) are met by
ATP.
IMP dehydrogenase is inhibited byGMP.
Reciprocal usage of energy ensures a
balanceof rate of synthesis of AMP &
GMP.
(When ATP is sufficient in cell, GMP will be
synthesised & when GTP is sufficient, AMPwill
besynthesised)
Adenosylsuccinate
synthetase
IMP
dehydrogenase

17. 4.1.3Conversion of GMP & AMPto GTP &ATP
•AMP and GMP are converted to diphosphates in reactionscatalyzed
by adenylate kinase and guanylate kinase,respectively.
adenylatekinase
AMP+ATP 2ADP
nucleosidediphosphate
kinase
ATP
Adenylate kinase is particularly active in liver andmuscle
guanylatekinase nucleosidediphosphate
kinase
GMP+ATP GDP+ADP GTP
•Conversion of nucleoside diphosphates to nucleoside triphosphatesis
catalyzed by nucleoside diphosphate kinase.
•The reaction is reversible, thus providing a way to make bothATP
andGTP.

18. 4.3SALVAGE PATHWAY FOR SYNTHESIS OFPURINES
•Preformed nucleotides can be recycledby salvagepathways.
•Purine nucleotides are synthesised preferentially by salvagepathway
•Purines from the normal turnover of cellular NAsor from the dietare not
degraded.
•“Salvage pathways"utilizes
i)the bases; hypoxanthine, guanine and adenine as substrates&
ii)the preformed nucleosides as thesubstrates
•There is specificity with respect to the base or nucleoside being"salvaged."
•The "salvage" of bases requires 2 distinct phospho ribosyltransferases.
1.Adenine phosphoribosyltransferase(APRTase)catalyses
Adenine+PRPP AMP+PPi
2.Hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) catalyses
thereactions
Hypoxanthine+PRPP
Guanine +PRPP
IMP+
GMP+
PPi and
PPi
•A deficiency of HGPRTase causes the Lesch-Nyhansyndrome.
1.
2.

19. 19
HMPShunt
SalvagePathway
De NovoSynthesis
Nucleotides
DNA,RNA
PRPPPurine,Pyrimidines
Ribose 5Phosphate
PRPPSynthetase ATP

20. 4.2Regulation of Denovo biosynthesis of purinenucleotides
1.Synthesis PRPP byPRPP
synthetase,
-activated byPi
-inhibited by purinenucleoside
di-andtriphosphates.
2.IMPdehydrogenaseistherate-
limitingenzyme-GMPactsasa
competitiveinhibitor.
3.Adenylosuccinate synthetase
is therate-limiting-AMP actsas
a competitiveinhibitor.
GMP AMP
2.
3.

21. Regulation of Denovo biosynthesis of purine nucletidescont…
4.The formation of 5-phospho
ribosyl amine (PRA) fromPRPP
and glutamine, catalysed by
glutamine PRPP
amidotransferase (GPA), is the
committed stepin IMPformation;
-rate-limiting
-regulated allosterically by the
end products -IMP, GMP &AMP
(negativeeffectors)
-PRPP is a positiveeffector.
GPA monomer isenzymatically
active.
In the presence of IMP, AMP, or
GMP, the enzyme forms adimer
that is much lessactive.
GlutaminePRPP
amidotranferase
IMPdehydrogenase
Adenylosuccinate
synthetase
glutamine PRPP
amidotransferase
(PRA)

22. 22
5.Catabolism of purinenucleotides to uric
acid
Adenosine
Inosine
Guanosine
Guanine
Xanthine
AMP GMP
Nucleotidase
Nucleotidase
Xanthineoxidase
Adenosinedeaminase
Guanine
deaminase
Xanthineoxidase
Purine nucleosidephosphorylase
UricAcid
(2,6,8-trioxypurine)
NH
4
Pi
Ribose-1-P
Hipoxanthine
NH4
H
2O
Pi
Xanthine oxidase inhibitedby
allopurinol

23. 6.Chemistry of uricacid
(a)Ionisation of uricacid
Both N-9(pK1=5.75) and N-3 (pK2 = 10.3) can ionise, but
ionisation of N-3 is not important since pK2 for the second protonis
10.3, a value well above that of physiologicfluids.
Urate
-1
ion Urate
-2
ion
(b) Solubility inwater
Ionicform(urates)are far more water-soluble than uricacid.
Solubility ofuricacid=6.5mg/dl
Solubility ofurateion=120mg/dl
Forminplasma-monosodiumurate
(2,6,8-trioxypurine)

24. Chemistry ofuricacidcont….
Forminurine-depends on pH ofurine
pH5.7
pH4.7
Uric acid (50 %) & monosodium urate (50%)
Uric acid (90 %) & monosodium urate (10%)
Sex Children Adults
Male
Female
2.0 -5.5mg/dl
3.5 -7.0 mg/dl (0.2 -0.42mmol/L)
2.6 -6.0mg/dl
(c).NormalValues
Uric acid is formed in 3ways;
i.by de novosynthesis
ii.by the metabolism of endogenous DNA, RNA and other purine containing
molecules such asATP
iii.by the breakdown of dietary nucleicacids.

25. 7.Hyperuricaemia andgout
(Read Kumar &Clark5
thEdn
Pg552-554)
•Hyperuricemia;
•The catabolism of the purines, A& G,produces uricacid.
Condition where the serum uric acid concentration exceeds 7.0mg/dl
(> 420umol/L).
Exceeds two SD from themean
(420 µmol/L in males, 360 µmol/L infemales).
•Uric acid levels in the blood depend on the balancebetween;
–purinesynthesis
–the ingestion of dietary purines, and
–the excretion of urate by the kidney and intestine(uricolysis).
•The body pool is about 1000 mg and 60% is turned overdaily.
•but ranges from 2000 to 4000 mg in gouty patients without tophiand
•may be as high as 30,000 mg in patients with severe tophaceousgout.

26. Normal balance of uric acidpool
bacteria-uricolysis,
leucocyteuricolysis
10 % ofthat
filtered by
glomerulus
Smalleramounts

27. (i)Common Causes ofhyperuricaemia

28. 8.Hyperuricaemia causes 4 clinical
syndromes;
Gout is characterised by hyperuricaemia, but only somewith
hyperuricaemia devolopgout.
1.Acute urate synovitis-Gout:
Triggered by the tissue deposition of sodium urate crystalswhich
cause an inflammatoryresponse
Sudden agonizing pain, swellingand rednessof the first MTP jointin
middle-agedmales.
•Attack may be precipitated by too much foodor alcohol,dehydration
or by starting adiuretic.
2. Chronic polyarticular gout
Unusual, except in elderly peopleon;
long-standing diuretictreatment,
in renal failure,or
in men who have been started on treatment with allopurinoltoosoon
after an acuteattack.

29. Hyperuricaemiacauses 4clinicalsyndromescont…
3. Chronic tophaceous gout(TG)
Sodium urate forms smooth white
deposits (tophi) in skin andaround
joints (Fig4).
They may occur on the ear lobe, the
fingersor the Achilles tendon.Large
deposits can alsoulcerate.
There is chronic joint pain&sometimes
superimposed acute goutyattacks.
•TG is often associated with renal
impairmentand/or the long-termuse
ofdiuretics.
4.Urate renal stoneformation
(Read KumarClark)

30. 30
Hyperurisaemia
Formation of monosodium urate crystalin sinovial fluid
Phagocytosisof crystal by polymorphonuclearleucocytes
Monosodium urate crystal causesinflammation
Secretionof chemotactic factor and enzyme inlysosome
Entry of moreleucocytes
Celldamage
Capillarypermeability
increases
InflammationInflammation

31. 9.Diagnosis &Treatment
Diagnosisofgout
1.Joint fluidmicroscopy:by observing joint fluid under apolarizing
light microscope for crystals ofurate;
-needle-shaped,
-intensively negatively birefringentcrystals
-crystals appear yellow when their long axis is parallel to theplane
of polarized light and blue when perpendicular toit.
2.Serum urate; usually raised 10mg/dl (> 600 umol/L), normal<400
Treatment
a) The use of NSAIDs in high doses rapidly reduces the pain & swelling;
naproxane, diclofenac, indomethacin-no direct effect on the serumurate
level.
•Alternative drug for renal impaired patients; colchicine,corticosteroids
b). Drugs forhyperuricaemia
Eg. probenecid promotes urateexcretion
•Allopurinol (300mg) -inhibitorofxanthineoxidase
•A diet low in purines and noalcohol.

33. Mechanism of action ofallopurinol
•Normally, Hypoxanthine is oxidised to xanthine by xanthine oxidase(xo)
and then to uricacid.
•Allopurinol, an analoque of hypoxanthine, is converted toalloxanthine.
•Alloxanthine binds tightly to the active site in xanthineoxidase-
suicide inhibition. Uric acid is notformed.
•Hypoxanthine and xanthine levels increase and areexcreted.

34. Inhibition of xanthine oxidase (XO) by alloxanthine is the mechanism involved
in allopurinol treatment ofgout.
Uricase is missing in humans, but is commonly used for measurement of
serum uric acid levels inhumans.

35. 35
5.Catabolism of purinenucleotides to uric
acid
Adenosine
Inosine
Guanosine
AMP GMP
Nucleotidase
Guanine
UricAcid
(2,6,8-trioxypurine)
Xanthine
Xanthineoxidase
Nucleotidase
Adenosinedeaminase
GuaninedeaminaseXanthineoxidase
Purine nucleosidephosphorylase
Ribose-1-P
Hipoxanthine
H
2O
Pi
NH
4
Pi
NH4
Xanthine oxidase inhibitedby
allopurinol

36. Questions
1.In addition to its activity as xanthine oxidase inhibitor,
what other activities of allopurinol might contribute to its
efficacy for treatment ofgout.
2.Describe the biochemical basis for the elevatedpurine
levels in Lesch-Nyhanpatients.
3.Name one example of purine-based anti-metabolite &
explain its mechanism ofaction

37. Reference
1.Lippincott’sBiochemistry2nd Edition Pgs; 343-355
2.Devlin, 5th Edition, pages; 826-858
3.BiochemistrybyLubertStryer4th Edition,pages;739-
759
4.Marks, Marks, Smith-Basic Medical Biochem. Pg630-6
26
th5.Harper’sIllustrated Pg293-302
6.http://web.indstate.edu/thcme/mwking/nucleotide-
metabolism.html
http://www.namrata.co/case-study-gout/