Bpt 114. purine and pyrimidine metabolism

Metabolism

1. Purine and Pyrimidine metabolism
BPT 114: DIGESTIVE, REPRODUCTIVE,
ENDOCRINE AND URINARY
SYSTEM
LECTURER;
Dr. Geoffrey K. Maiyoh
Department of Medical Biochemistry
GKM/MUSOM/BPT 114:DIG.END.REP.URISYS.2014

2. •Purines and pyrimidines are Nitrogen containing
heterocyclic compounds whose rings contain Carbon
and Nitrogen.
•The planar character of purines and pyrimidines
facilitates their close association or stacking which
stabilizes double stranded DNA.
•They are also refereed to as Nitrogenous Bases and
are a major components of nucleotides that build
DNA and RNA
PURINES AND PYRIMIDINES
GKM/MUSOM/BPT 114:DIG.END.REP.URISYS.2014

3. Biological functions of nucleotides
    Building blocks of nucleic acids (DNA and RNA).
     Involved in energy storage, muscle contraction, 
active transport, maintenance of ion gradients.
      Activated intermediates in biosynthesis 
(e.g. UDP-glucose, S-adenosylmethionine).
      Components of coenzymes (NAD
+
, NADP
+
, FAD, 
FMN, and CoA)
      Metabolic regulators:
a.      Second messengers (cAMP, cGMP)
b.      Phosphate donors in signal transduction (ATP) 
c.       Regulation of some enzymes via adenylation and 
   uridylylation 
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4. Nitrogenous Bases
•Planar, aromatic, and heterocyclic compounds
•Derived from purine or pyrimidine
•Numbering of bases is “unprimed”
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5. Nucleic Acid Bases
Purines
Pyrimidines
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6. Sugars
•Pentoses (5-C sugars)
•Numbering of sugars is “primed”
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7. Sugars
D-Ribose and 2’-Deoxyribose
*Lacks a 2’-OH group
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8. Nucleosides
•Result from linking one of the sugars with a
purine or pyrimidine base through an N-
glycosidic linkage
–Purines bond to the C1’ carbon of the sugar at
their N9 atoms
–Pyrimidines bond to the C1’ carbon of the
sugar at their N1 atoms
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9. Nucleosides
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10. Phosphate Groups
•Mono-, di- or triphosphates
•Phosphates can be bonded to either C3 or
C5 atoms of the sugar
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11. Nucleotides
•Result from linking one or more phosphates with a
nucleoside onto the 5’ end of the molecule through
esterification
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12. Nucleotides
•RNA (ribonucleic acid) is a polymer of
ribonucleotides
•DNA (deoxyribonucleic acid) is a polymer
of deoxyribonucleotides
•Both deoxy- and ribonucleotides contain
Adenine, Guanine and Cytosine
–Ribonucleotides contain Uracil
–Deoxyribonucleotides contain Thymine
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13. Nucleotides
•Monomers for nucleic acid polymers
•Nucleoside Triphosphates are important
energy carriers (ATP, GTP)
•Important components of coenzymes
–FAD, NAD
+
and Coenzyme A
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14. Naming Conventions
•Nucleosides:
–Purine nucleosides end in “-sine”
•Adenosine, Guanosine
–Pyrimidine nucleosides end in “-dine”
•Thymidine, Cytidine, Uridine
•Nucleotides:
–Start with the nucleoside name from above and
add “mono-”, “di-”, or “triphosphate”
•Adenosine Monophosphate, Cytidine Triphosphate,
Deoxythymidine Diphosphate
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15. Nucleotides
-O
O
H(OH)
HH
HH
O
OP
O
O-
Purine or
Pyrimidine
Base
Phosphate
Pentose sugar
Nucleoside
Nucleotide
1'
2'3'
4'
5'
b-glycosidic bond
RNA- ribose (R)
DNA – deoxyribose (dR)
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16. N
N
N
N
H
NH
2
N
NH
N
H
N
H
O
O
N
N
H
NH
2
O
H
3C
NH
N
H
O
O
Xanthine
Adenine (A)
Thymine (T)Cytosine (C)
NH
N
H
O
O
Uracil (U)
N
N
N
N
H
Purine
N
N
H
Pyrimidine
1
2
3
4
5
6
3
2
1
6
4
5
7
8
9
N
NH
N
N
H
O
NH
2
Guanine (G)
N
NH
N
N
H
O
Hypoxanthine
Nucleobase structures
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17. Hypoxanthine Inosine Inosinate (IMP)
Xanthine Xanthosine Xanthylate (XMP)
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18. Two major routes for nucleotide
biosynthesis
dNTPs
dNTPs
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19. Synthesis Pathways
•For both purines and pyrimidines there are two means
of synthesis (often regulate one another)
–de novo (from basic metabolites)
–salvage (recycle from pre-existing nucleotides)
Salvage Pathwayde novo Pathway
GKM/MUSOM/BPT 114:DIG.END.REP.URISYS.2014

20. Purine nucleotides
–Purines are not initially synthesized as free bases
–First purine derivative formed is Inosine Mono-phosphate
(IMP)
•The purine base is hypoxanthine
•AMP and GMP are formed from IMP
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21. Purine Nucleotides
•Get broken down into Uric Acid (a purine)
Buchanan (mid 1900s) showed where purine ring
components came from:
N
1
: Aspartate Amine
C2, C8: Formate
N3, N9: Glutamine
C4, C5, N7: Glycine
C6: Bicarbonate Ion
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22. GKM/MUSOM/BPT
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23. Purine Nucleotide Synthesis
OH
H
H
CH
2
OH OH
H H
O
a
O
2-
O
3P
a-D-Ribose-5-Phosphate (R5P)
O
H
H
CH
2
OH OH
H H
O
a
O
2-
O
3P
5-Phosphoribosyl-a-pyrophosphate (PRPP)
P
O
O
OP
O
O
O
ATP
AMP
Ribose
Phosphate
Pyrophosphokinase
H
NH
2
H
CH
2
OH OH
H H
O
b
O
2-
O
3P
b-5-Phosphoribosylamine (PRA)
Amidophosphoribosyl
Transferase
Glutamine
+ H
2O
Glutamate
+ PP
i
H
NH
H
CH
2
OH OH
H H
O
O
2-
O
3P
CO
H
2CNH
2
Glycinamide Ribotide (GAR)
GAR Synthetase
Glycine
+ ATP
ADP
+ P
i
H
2C
C
NH
O
CH
H
N
O
Ribose-5-Phosphate
Formylglycinamide ribotide (FGAR)
H
2C
C
NH
O
CH
H
N
HN
Ribose-5-Phosphate
Formylglycinamidine ribotide (FGAM)
THFN
10
-Formyl-THF
GAR Transformylase
ATP +
Glutamine +
H
2O
ADP +
Glutamate + P
i
FGAM
Synthetase
HC
C
N
CH
N
H
2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole Ribotide (AIR)
ATP
ADP + P
i
AIR
Synthetase
C
C
N
CH
N
H
2N
OOC
Ribose-5-Phosphate
4
5
Carboxyamidoimidazole Ribotide (CAIR)
ATP
+HCO
3
ADP + P
i
AIR
Car boxylase
Aspartate
+ ATP
ADP
+ P
i
SAICAR Synthetase
Adenylosuccinate
Lyase
Fumarate
C
C
N
CH
N
NH
Ribose-5-Phosphate
4
5
5-Formaminoimidazole-4-carboxamide
ribotide (FAICAR)
C
H
2N
O
C
H
O
C
C
N
CH
N
H
2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-carboxamide
ribotide (AICAR)
C
H
2N
O
C
C
N
CH
N
H
2N
C
N
H
O
HC
COO
CH
2
COO
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-(N-succinylocarboxamide)
ribotide (SAICAR)
THF
AICAR
Transformylase
N
10
-Formyl-
THF
Inosine Monophosphate (IMP)
HN
HC
N
C
C
C
N
CH
N
O
4
5
HH
CH2
OH OH
H H
O
O
2-
O
3P
IMP
Cyclohydrolase
H
2O

24. Purine Nucleotide Synthesis
at a Glance
•ATP is involved in 6 steps
•PRPP in the first step of Purine synthesis is also a precursor for
Pyrimidine Synthesis, His and Trp synthesis
–Role of ATP in first step is unique– group transfer rather than
coupling
•In second step, C1 notation changes from a to b (anomers
specifying OH positioning on C1 with respect to C4 group)
•In step 2, PPi is hydrolyzed to 2Pi (irreversible, “committing” step)
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25. de novo Synthesis and regulation
•Committed step: This is the point of no return
–Occurs early in the biosynthetic pathway
–Often regulated by final product (feedback
inhibition)
X
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26. •Hydrolyzing a phosphate from ATP is relatively easy
DG°’= -30.5 kJ/mol
–If exergonic reaction released energy into cell as heat energy,
wouldn’t be useful
–Must be coupled to an endergonic reaction
•When ATP is a reactant:
–Part of the ATP can be transferred to an acceptor: Pi, PPi, adenyl,
or adenosinyl group
–ATP hydrolysis can drive an otherwise unfavorable reaction
(synthetase; “energase”)
Coupling of Reactions
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27. Substrate Channeling
•Substrate channeling is the process of direct
transfer of an intermediate between the active
sites of two enzymes that catalyze sequential
reactions in a biosynthetic pathway.
•The active sites can be located either on
separate domains in a multifunctional enzyme
or on separate subunits in a multienzyme
complex.
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28. Purine Biosynthetic Pathway
•Channeling of some reactions on pathway
organizes and controls processing of substrates to
products in each step
–Increases overall rate of pathway and protects
intermediates from degradation
•In animals, IMP synthesis pathway shows
channeling at:
–Reactions 3, 4, 6
–Reactions 7, 8
–Reactions 10, 11
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29. IMP Conversion to
AMP and GMP

30. Regulatory Control of Purine Nucleotide Biosynthesis
•GTP is involved in AMP synthesis and ATP is involved in
GMP synthesis (reciprocal control of production)
•Women in love with Catholic priests are calling for the
Pope to change celibacy laws (and why we don't blame
them) http://www.christiantoday.com/article/dear.francis.let.our.lovers.marry/37536.htm
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"We humbly place our suffering at your feet in
the hope that something may change, not just
for us, but for the good of the entire Church.
We love these men, they love us, and in most
cases, despite all efforts to renounce it, one
cannot manage to give up such a solid and
beautiful bond.“..Christian today, 10
th
June,
2014

31. Regulatory Control of Purine Biosynthesis
•Above the level of IMP production:
–Independent control
–Synergistic control
–Feed forward activation by PRPP
•Below level of IMP production
–Reciprocal control
Overall
•Total amounts of purine nucleotides is controlled
•Relative amounts of ATP, GTP controlled
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32. Purine Catabolism and Salvage
•All purine degradation leads to uric acid (but it might not
stop there)
•Ingested nucleic acids are degraded to nucleotides by
pancreatic nucleases, and intestinal phosphodiesterases in
the intestine
•Group-specific nucleotidases and non-specific
phosphatases degrade nucleotides into nucleosides
–Direct absorption of nucleosides
–Further degradation
Nucleoside + H2O  base + ribose (nucleosidase)
Nucleoside + P
i
 base + r-1-phosphate (n. phosphorylase)
NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND
EXCRETED.
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33. GMO phobia
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We can not even absorb whole nucleotides, how then do we absorb
a gene???

34. Intracellular Purine Catabolism
•Nucleotides broken into nucleosides by action of
5’-nucleotidase (hydrolysis reactions)
•Purine nucleoside phosphorylase (PNP)
–Inosine  Hypoxanthine
–Xanthosine  Xanthine
–Guanosine  Guanine
–Ribose-1-phosphate splits off
•Can be isomerized to ribose-5-phosphate
•Adenosine is deaminated to Inosine (ADA)
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35. Intracellular Purine Catabolism
•Xanthine is the point of convergence for the
metabolism of the purine bases
•Xanthine  Uric acid
–Xanthine oxidase catalyzes two reactions
•Purine ribonucleotide degradation pathway
is same for purine deoxyribonucleotides
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36. Major pathways of purine
catabolism in animals.
ADA

37. Uric Acid Excretion
•Humans – excreted into urine as insoluble
crystals
•Birds, terrestrial reptiles, some insects –
excrete insoluble crystals in paste form
–Excess amino N converted to uric acid
•(conserves water)
•Others – further modification :
Uric Acid  Allantoin  Allantoic Acid  Urea 
Ammonia
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38. Purine Salvage
•Adenine phosphoribosyl transferase (APRT)
Adenine + PRPP  AMP + PPi
•Hypoxanthine-Guanine phosphoribosyl transferase
(HGPRT)
Hypoxanthine + PRPP  IMP + PPi
Guanine + PRPP  GMP + PPi
(NOTE: THESE ARE ALL REVERSIBLE REACTIONS)
AMP,IMP,GMP do not need to be resynthesized de
novo !
GKM/MUSOM/BPT
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39. Gout
•Impaired excretion or overproduction of uric
acid
•Uric acid crystals precipitate into joints (Gouty
Arthritis), kidneys, ureters (stones)
•Lead impairs uric acid excretion – lead
poisoning from pewter drinking goblets
–Fall of Roman Empire?
•Xanthine oxidase inhibitors inhibit production
of uric acid, and treat gout
•Allopurinol treatment – hypoxanthine analog
that binds to Xanthine Oxidase to decrease uric
acid production
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40. GKM/MUSOM/BPT
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41. ALLOPURINOL IS A XANTHINE OXIDASE
INHIBITOR
A SUBSTRATE ANALOG IS CONVERTED TO AN
INHIBITOR, IN THIS CASE A “SUICIDE-INHIBITOR”
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42. Pyrimidine Ribonucleotide Synthesis
• Uridine Monophosphate (UMP) is
synthesized first
–CTP is synthesized from UMP
•Pyrimidine ring synthesis completed first;
then attached to ribose-5-phosphate
N1, C4, C5, C6 : Aspartate
C2 : HCO3
-
N3 : Glutamine amide Nitrogen
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44. 2 ATP + HCO
3
-
+ Glutamine + H
2O
CO
O PO
3
-2
NH
2
Carbamoyl Phosphate
NH
2
C
N
H
CH
CH
2
C
COO
O
HO
O
Carbamoyl Aspartate
HN
C
N
H
CH
CH
2
C
COO
O
O
Dihydroorotate
HN
C
N
H
C
CH
C
COO
O
O
Orotate
HN
C
N
C
CH
C
COO
O
O
HH
CH
2
OH OH
H H
O
O
2-
O
3P
b
Orotidine-5'-monophosphate
(OMP)
HN
C
N
CH
CH
C
O
O
HH
CH
2
OH OH
H H
O
O
2-
O
3P
b
Uridine Monophosphate
(UMP)
2 ADP +
Glutamate +
P
i
Carbamoyl
Phosphate
Synthetase II
Aspartate
Transcarbamoylase
(ATCase)
Aspartate
P
i
H
2O
Dihydroorotase
Quinone
Reduced
Quinone
Dihydroorotate
Dehydrogenase
PRPP PP
i
Orotate Phosphoribosyl
Transferase
CO
2
OMP
Decarboxylase
Pyrimidine Synthesis
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53

45. UMP Synthesis Overview
•2 ATPs needed: both used in first step
–One transfers phosphate, the other is hydrolyzed to ADP and Pi
•2 condensation rxns: form carbamoyl aspartate and
dihydroorotate (intramolecular)
•Dihydroorotate dehydrogenase is an intra-mitochondrial
enzyme; oxidizing power comes from quinone reduction
•Attachment of base to ribose ring is catalyzed by OPRT;
PRPP provides ribose-5-P
–PP
i
splits off PRPP – irreversible
•Channeling: enzymes 1, 2, and 3 on same chain; 5 and 6 on
same chain
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46. UMP  UTP and CTP
•Nucleoside monophosphate kinase catalyzes
transfer of Pi to UMP to form UDP; nucleoside
diphosphate kinase catalyzes transfer of Pi from
ATP to UDP to form UTP
•CTP formed from UTP via CTP Synthetase
driven by ATP hydrolysis
–Glutamine provides amide nitrogen for C4 in
animals
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47. GKM/MUSOM/BPT
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48. Regulatory Control of Pyrimidine
Synthesis
•Differs between bacteria and animals
–Bacteria – regulation at ATCase rxn
•Animals – regulation at carbamoyl phosphate synthetase
II
–UDP and UTP inhibit enzyme; ATP and PRPP activate it
–UMP and CMP competitively inhibit OMP Decarboxylase
*Purine synthesis inhibited by ADP and GDP at ribose
phosphate pyrophosphokinase step, controlling level of
PRPP  also regulates pyrimidines
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49. Orotic Aciduria
•Caused by defect in protein chain with enzyme
activities of last two steps of pyrimidine
synthesis
•Increased excretion of orotic acid in urine
•Symptoms: retarded growth; severe anemia
•Only known inherited defect in this pathway
(all others would be lethal to fetus)
•Treat with uridine/cytidine
•IN-CLASS QUESTION: HOW DOES URIDINE
AND CYTIDINE ADMINISTRATION WORK TO
TREAT OROTIC ACIDURIA?
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50. How does UMP/UTP Cure
Orotic Aciduria?
UMP
Synthetase
X
Carbamoyl
Phosphate
Orotate
Feedback
Inhibition
•Disease (-UMP)
–No UMP/excess orotate
•Disease (+UMP)
–Restore depleted UMP
–Downregulate pathway via feedback inhibition (Less orotate)
55

51. Degradation of Pyrimidines
•CMP and UMP degraded to bases similarly
to purines
–Dephosphorylation
–Deamination
–Glycosidic bond cleavage
•Uracil reduced in liver, forming b-alanine
–Converted to malonyl-CoA  fatty acid
synthesis for energy metabolism
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52. Thank you for your attention
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