Bpt 114. purine and pyrimidine metabolism

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

• 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

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

Nitrogenous Bases
• Planar, aromatic, and heterocyclic compounds
• Derived from purine or pyrimidine
• Numbering of bases is “unprimed”
GKM/MUSOM/BPT
114:DIG.END.REP.URISYS.2014

Nucleic Acid Bases
Purines Pyrimidines
GKM/MUSOM/BPT

Sugars
• Pentoses (5-C sugars)
• Numbering of sugars is “primed”
GKM/MUSOM/BPT

Sugars
D-Ribose and 2’-Deoxyribose
*Lacks a 2’-OH group
GKM/MUSOM/BPT

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
GKM/MUSOM/BPT

Nucleosides
GKM/MUSOM/BPT

Phosphate Groups
• Mono-, di- or triphosphates
• Phosphates can be bonded to either C3 or
C5 atoms of the sugar
GKM/MUSOM/BPT

Nucleotides
• Result from linking one or more phosphates with a
nucleoside onto the 5’ end of the molecule through
esterification
GKM/MUSOM/BPT

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
GKM/MUSOM/BPT

Nucleotides
• Monomers for nucleic acid polymers
• Nucleoside Triphosphates are important
energy carriers (ATP, GTP)
• Important components of coenzymes
– FAD, NAD+
and Coenzyme A
GKM/MUSOM/BPT

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
GKM/MUSOM/BPT

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'
β-glycosidic bond
RNA- ribose (R)
DNA – deoxyribose (dR)

N
N
NN
H
NH2
N
NH
N
H
N
H
O
O
N
N
H
NH2
O
H3C
NH
N
H
O
O
XanthineAdenine (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
NH2
Guanine (G)
N
NH
N
N
H
O
Hypoxanthine
Nucleobase structures

Hypoxanthine Inosine Inosinate (IMP)
Xanthine Xanthosine Xanthylate (XMP)
GKM/MUSOM/BPT

Two major routes for nucleotide
biosynthesis
dNTPs
dNTPs

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

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
GKM/MUSOM/BPT

Purine Nucleotides
• Get broken down into Uric Acid (a purine)
Buchanan (mid 1900s) showed where purine ring
components came from:
N1: Aspartate Amine
C2, C8: Formate
N3, N9: Glutamine
C4, C5, N7: Glycine
C6: Bicarbonate Ion
GKM/MUSOM/BPT

GKM/MUSOM/BPT

Purine Nucleotide Synthesis
OH
H
H
CH2
OH OH
H H
O
α
O2-
O3P
α-D-Ribose-5-Phosphate (R5P)
O
H
H
CH2
OH OH
H H
O α
O2-
O3P
5-Phosphoribosyl-α-pyrophosphate (PRPP)
P
O
O
O P
O
O
O
ATP
AMP
Ribose
Phosphate
Pyrophosphokinase
H
NH2
H
CH2
OH OH
H H
O
β
O2-
O3P
β-5-Phosphoribosylamine (PRA)
Amidophosphoribosyl
Transferase
Glutamine
+ H2O
Glutamate
+ PPi
H
NH
H
CH2
OH OH
H H
O
O2-
O3P
CO
H2C NH2
Glycinamide Ribotide (GAR)
GAR Synthetase
Glycine
+ ATP
ADP
+ Pi
H2C
C
NH
O
CH
H
N
O
Ribose-5-Phosphate
Formylglycinamide ribotide (FGAR)
H2C
C
NH
O
CH
H
N
HN
Ribose-5-Phosphate
Formylglycinamidine ribotide (FGAM)
THFN10
-Formyl-THF
GAR Transformylase
ATP +
Glutamine +
H2O
ADP +
Glutamate + Pi
FGAM
Synthetase
HC
C
N
CH
N
H2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole Ribotide (AIR)
ATP
ADP + Pi
AIR
Synthetase
C
C
N
CH
N
H2N
OOC
Ribose-5-Phosphate
4
5
Carboxyamidoimidazole Ribotide (CAIR)
ATP
+HCO3
ADP + Pi
AIR
Car boxylase
Aspartate
+ ATP
ADP
+ Pi
SAICAR Synthetase
Adenylosuccinate
Lyase
Fumarate
C
C
N
CH
N
NH
Ribose-5-Phosphate
4
5
5-Formaminoimidazole-4-carboxamide
ribotide (FAICAR)
C
H2N
O
C
H
O
C
C
N
CH
N
H2N
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-carboxamide
ribotide (AICAR)
C
H2N
O
C
C
N
CH
N
H2N
C
N
H
O
HC
COO
CH2
COO
Ribose-5-Phosphate
4
5
5-Aminoimidazole-4-(N-succinylocarboxamide)
ribotide (SAICAR)
THF
AICAR
Transformylase
N10
-Formyl-
THF
Inosine Monophosphate (IMP)
HN
HC
N
C
C
C
N
CH
N
O
4
5
HH
CH2
OH OH
H H
OO2-
O3P
IMP
Cyclohydrolase
H2O

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 α to β (anomers
specifying OH positioning on C1 with respect to C4 group)
• In step 2, PPi is hydrolyzed to 2Pi (irreversible, “committing” step)
GKM/MUSOM/BPT

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
GKM/MUSOM/BPT

• Hydrolyzing a phosphate from ATP is relatively easy
∆G°’= -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
GKM/MUSOM/BPT

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.
GKM/MUSOM/BPT

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
GKM/MUSOM/BPT

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
GKM/MUSOM/BPT
"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, 10th
June,
2014

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
GKM/MUSOM/BPT

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 + Pi  base + r-1-phosphate (n. phosphorylase)
NOTE: MOST INGESTED NUCLEIC ACIDS ARE DEGRADED AND
EXCRETED.
GKM/MUSOM/BPT

GMO phobia
GKM/MUSOM/BPT
We can not even absorb whole nucleotides, how then do we absorb
a gene???

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)
GKM/MUSOM/BPT

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
GKM/MUSOM/BPT

Major pathways of purine
catabolism in animals.
ADA

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
GKM/MUSOM/BPT

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

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
GKM/MUSOM/BPT

ALLOPURINOL IS A XANTHINE OXIDASE
INHIBITOR
A SUBSTRATE ANALOG IS CONVERTED TO AN
INHIBITOR, IN THIS CASE A “SUICIDE-INHIBITOR”
GKM/MUSOM/BPT

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
GKM/MUSOM/BPT

2 ATP + HCO3
-
+ Glutamine + H2O
CO
O PO3
-2
NH2
Carbamoyl Phosphate
NH2
C
N
H
CH
CH2
C
COO
O
HO
O
Carbamoyl Aspartate
HN
C
N
H
CH
CH2
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
CH2
OH OH
H H
O
O2-
O3P
β
Orotidine-5'-monophosphate
(OMP)
HN
C
N
CH
CH
C
O
O
HH
CH2
OH OH
H H
O
O2-
O3P
β
Uridine Monophosphate
(UMP)
2 ADP +
Glutamate +
Pi
Carbamoyl
Phosphate
Synthetase II
Aspartate
Transcarbamoylase
(ATCase)
Aspartate
Pi
H2O
Dihydroorotase
Quinone
Reduced
Quinone
Dihydroorotate
Dehydrogenase
PRPP PPi
Orotate Phosphoribosyl
Transferase
CO2
OMP
Decarboxylase
Pyrimidine Synthesis
GKM/MUSOM/BPT
53

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
– PPi splits off PRPP – irreversible
• Channeling: enzymes 1, 2, and 3 on same chain; 5 and 6 on
same chain
GKM/MUSOM/BPT

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 C4in
animals
GKM/MUSOM/BPT

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
GKM/MUSOM/BPT

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?
GKM/MUSOM/BPT

How does UMP/UTP Cure
Orotic Aciduria?
UMP
Synthetase
XCarbamoyl
Phosphate
Orotate
Feedback
Inhibition
• Disease (-UMP)
– No UMP/excess orotate
• Disease (+UMP)
– Restore depleted UMP
– Downregulate pathway via feedback inhibition (Less orotate)
55

Degradation of Pyrimidines
• CMP and UMP degraded to bases similarly
to purines
– Dephosphorylation
– Deamination
– Glycosidic bond cleavage
• Uracil reduced in liver, forming β-alanine
– Converted to malonyl-CoA  fatty acid
synthesis for energy metabolism
GKM/MUSOM/BPT

Thank you for your attention
GKM/MUSOM/BPT

Bpt 114. purine and pyrimidine metabolism

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