BIOMEDIC I-BIOCHEMISTRY

DEPARTMENT OF BIOCHEMISTRY.
FACULTY OF MEDICINE
UNIVERSITAS HASANUDDIN
 TOPIC

NUCLEOTIDE PURINE AND PIRIMIDINE

METABOLISM.
 Content :
Introduction.
Structure of nucleoside and nucleotide.
Purine nucleotide synthesis.
Regulation control of Purine nucleotide synthesis.
Purine Catabolism and Salvage pathway.
Uric acid synthesis.
Pyrimidine Ribonucleotide Synthesis.
Regulatory Control of Pyrimidine Synthesis.
Degradation of Pyrimidines
 INTRODUCTION
Purine is a heterocyclic aromatic organic compound that
consists of a pyrimidine ring fused to an imidazole ring.
It is water-soluble.
Purine also gives its name to the wider class of
molecules, purines, which include substituted purines
and their tautomers.
They are the most widely occurring nitrogen-containing
heterocycles in nature.
Purines are found in high concentration in meat and
meat products, especially internal organs such as liver
and kidney.
In general, plant-based diets are low in purines.
Examples of high-purine sources include: sweetbreads,
anchovies, sardines, liver, beef kidneys, brains, meat
extracts (e.g., Oxo, Bovril), herring, mackerel, scallops,
game meats, beer (from the yeast) and gravy.
Pyrimidine is an aromatic heterocyclic organic compound similar to
pyridine. One of the three diazines (six-membered heterocyclics with
two nitrogen atoms in the ring), it has the nitrogen atoms at positions
1 and 3 in the ring.
The other diazines are pyrazine (nitrogen atoms at the 1 and 4
positions) and pyridazine (nitrogen atoms at the 1 and 2 positions).
In nucleic acids, three types of nucleobases are pyrimidine
derivatives: cytosine (C), thymine (T), and uracil (U).
PURINES, PYRIMIDINES, NUCLEOSIDES,
& NUCLEOTIDES .

Nucleotides: Synthesis and

Degradation
 Nitrogenous Bases
Planar, aromatic, and heterocyclic
Derived from purine or pyrimidine
Numbering of bases is “unprimed”
 Nucleic Acid Bases
Purines Pyrimidines
 Sugars
Pentoses (5-C sugars)
Numbering of sugars is “primed”
 Sugars
D-Ribose and 2’-Deoxyribose

*Lacks a 2’-OH group

 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
Nucleosides
 Phosphate Groups
Mono-, di- or triphosphates

Phosphates can be bonded to either C3 or

C5 atoms of the sugar
 Nucleotides
Result from linking one or more phosphates
with a nucleoside onto the 5’ end of the
molecule through esterification
 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
 Nucleotides
Monomers for nucleic acid polymers
Nucleoside Triphosphates are important
energy carriers (ATP, GTP)
Important components of coenzymes
– FAD, NAD+ and Coenzyme A
 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
 Nucleosides

(1)

(9)
 Nucleotides
• Phosphate ester of nucleosides
RNA contains Ribose while DNA contains 2'deoxy-D-Ribose
Nucleoside: Nitrogenous base + ribose:
Ribonucleotides
Adenosine 5'-monophosphate, Adenylate, AMP
Guanosine 5'-monophosphate, Guanylate, GMP
Cytidine 5'-monophosphate, Cytidylate, CMP
Uridine 5'-monophosphate, Uridylate, UMP
Deoxyribonucleotides
Deoxythymidine 5' monophosphate,
Deoxythymidine, dTMP ……..
NMP=== Nucleoside mono phosphate.
Numbering of Ribose sugar is given 1’, 2’, …, 5’
 Unusual nucleotides
Modified nucleotides found in some viral DNA and in Transfer RNA. These
modifications include methylation, hydroxymethylation, glycosylation,
acetylation ..
 Other Functions of Nucleotides
• Nucleoside 5'-triphosphates are carriers of energy
• Bases serve as recognition units
• Cyclic nucleotides are signal molecules and regulators of
cellular metabolism and reproduction
• Structural component of some coenzymes, e.g CoA, FAD,
NADH, NADPH
• ATP is central to energy metabolism
• GTP drives protein synthesis
• CTP drives lipid synthesis
• UTP drives carbohydrate metabolism
De novo synthesis of purine nucleotides
The atoms of purine ring are contributed by a number of compounds including
amino acids (aspartic acid, glycine and glutamine) CO2, and tetrahydrofolate.
These compounds donates N and C to constructed Ribose 5-phosphate.

6
5 7
1

2 8
4 N10Formyl tetrahydrofolate
9
3
The order in which ring atoms are added is:

9 4 5 7 8 3 6 1 2

Glut Glycine For Glut CO2 Asp For

6
5 N 7
1 N
8
2
N N
4 9
3

Ribose-P
 Nucleotide Metabolism
PURINE RIBONUCLEOTIDES: formed de novo
– i.e., 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
 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
Biosynthesis Purine
Continued………
 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)
 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
IMP Conversion to AMP
IMP Conversion to GMP
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.
 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)
 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
Adenosine Degradation
 Xanthosine Degradation

• Ribose sugar gets recycled (Ribose-1-Phosphate  R-5-P )

– can be incorporated into PRPP (efficiency)
• Hypoxanthine is converted to Xanthine by Xanthine Oxidase
• Guanine is converted to Xanthine by Guanine Deaminase
• Xanthine gets converted to Uric Acid by Xanthine Oxidase
 Xanthine Oxidase
A homodimeric protein
Contains electron transfer proteins
– FAD
– Mo-pterin complex in +4 or +6 state
– Two 2Fe-2S clusters
Transfers electrons to O2  H2O2
– H2O2 is toxic
– Disproportionated to H2O and O2 by catalase
 THE PURINE NUCLEOTIDE CYCLE
AMP + H2O  IMP + NH4+ (AMP Deaminase)

IMP + Aspartate + GTP  AMP + Fumarate + GDP + Pi

(Adenylosuccinate Synthetase)

COMBINE THE TWO REACTIONS:

Aspartate + H2O + GTP  Fumarate + GDP + Pi + NH4+

The overall result of combining reactions is deamination of Aspartate to

Fumarate at the expense of a GTP
 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

Degradation of
Purine nucleotides
 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 !
 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
 ALLOPURINOL IS A XANTHINE OXIDASE
INHIBITOR

A SUBSTRATE ANALOG IS CONVERTED TO AN

INHIBITOR, IN THIS CASE A “SUICIDE-INHIBITOR”
Lesch-Nyhan Syndrome
 A defect in production or activity of
HGPRT
 Causes increased level of Hypoxanthine and
Guanine ( in degradation to uric acid)
 Also,PRPP accumulates
 stimulates production of purine nucleotides
(and thereby increases their degradation)
 Causes gout-like symptoms, but also
neurological symptoms  spasticity,
aggressiveness, self-mutilation
 First neuropsychiatric abnormality that
was attributed to a single enzyme
 Purine Autism
 25% of autistic patients may
overproduce purines
 To diagnose, must test urine over
24 hours
 Biochemical findings from this test
disappear in adolescence
 Must obtain urine specimen in
infancy, but it’s difficult to do!
• Pink urine due to uric acid crystals may
be seen in diapers
 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
 Pyrimidine Synthesis
O

2 ATP + HCO3- + Glutamine + H2O C

HN CH
2 ADP +
Carbamoyl O
Glutamate +
Phosphate C C
Pi
Synthetase II
C O N
HN CH PRPP PPi COO
2-
O3P O CH2
O
NH2
C C Orotate Phosphoribosyl H H b
N Transferase
O C O H H
H COO OH OH
O PO3-2 Orotidine-5'-monophosphate
Orotate
(OMP)
Carbamoyl Phosphate
Reduced
Aspartate Quinone OMP
Aspartate Dihydroorotate
Transcarbamoylase Decarboxylase
Dehydrogenase CO2
(ATCase)
Pi Quinone O

O C
O HN CH

C
HO C C CH
HN CH2 N
CH2 O
NH2 H2O
2-
C CH O3P O CH2
O
C CH O N H H b
Dihydroorotase
O N H COO H H
H COO OH
OH
Dihydroorotate
Carbamoyl Aspartate Uridine Monophosphate
(UMP)
 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
 OMP DECARBOXYLASE : THE MOST
CATALYTICALLY PROFICIENT ENZYME
 FINAL REACTION OF PYRIMIDINE PATHWAY
 ANOTHER MECHANISM FOR DECARBOXYLATION
 A HIGH ENERGY CARBANION INTERMEDIATE NOT
NEEDED
 NO COFACTORS NEEDED !
 SOME OF THE BINDING ENERGY BETWEEN OMP
AND THE ACTIVE SITE IS USED TO STABILIZE
THE TRANSITION STATE
• “PREFERENTIAL TRANSITION STATE BINDING”
 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
 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
 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?
 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
Degradation of pyrimidines
- Purines are not cleaved in human cell
- pyrimidines rings can be opened and degraded to highly
soluble structure, such as b-alanine and b-aminoisobutyrate
that can serve as precursors of acetyl CoA and succinyl CoA
- Pyrimidine can be salvaged and converted into nucleotides
by the enzyme Pyrimidine phosphoribosyltransferase and it
utilizes the PRPP
Deoxyribonucleotide Formation
 Purine/Pyrimidine degradation are the
same for ribonucleotides and
deoxyribonucleotides

 Biosynthetic pathways are only for

ribonucleotide production

 Deoxyribonucleotides are synthesized

from corresponding ribonucleotides
 Thymine Formation
 Formed by methylating deoxyuridine
monophosphate (dUMP)
 UTP is needed for RNA production, but
dUTP not needed for DNA
• If dUTP produced excessively, would cause
substitution errors (dUTP for dTTP)
 dUTP hydrolyzed by dUTPase
(dUTP diphosphohydrolase) to dUMP 
methylated at C5 to form dTMP
rephosphorylate to form dTTP
 Tetrahydrofolate (THF)
 Methylation of dUMP catalyzed by
thymidylate synthase
• Cofactor: N5,N10-methylene THF
 Oxidized to dihydrofolate
 Only known rxn where net oxidation state
of THF changes
 THF Regeneration:
DHF + NADPH + H+  THF + NADP+ (enzyme: dihydrofolate
reductase)

THF + Serine  N5,N10-methylene-THF + Glycine

(enzyme: serine hydroxymethyl transferase)
 Anti-Folate Drugs
 Cancer cells consume dTMP quickly for
DNA replication
• Interfere with thymidylate synthase rxn to
decrease dTMP production
 (fluorodeoxyuridylate – irreversible inhibitor) – also
affects rapidly growing normal cells (hair follicles,
bone marrow, immune system, intestinal mucosa)
 Dihydrofolate reductase step can be
stopped competitively (DHF analogs)
• Anti-Folates: Aminopterin, methotrexate,
trimethoprim
 ADENOSINE DEAMINASE DEFICIENCY

 IN PURINE DEGRADATION, ADENOSINE 

INOSINE
• ENZYME IS ADA
 ADA DEFICIENCY RESULTS IN SCID
• “SEVERE COMBINED IMMUNODEFICIENCY”
 SELECTIVELY KILLS LYMPHOCYTES
• BOTH B- AND T-CELLS
• MEDIATE MUCH OF IMMUNE RESPONSE
 ALL KNOWN ADA MUTANTS STRUCTURALLY
PERTURB ACTIVE SITE
 Adenosine Deaminase
CHIME Exercise: 2ADA
Enzyme catalyzing deamination of Adenosine to Inosine
a/b barrel domain structure
– “TIM Barrel” – central barrel structure with 8 twisted
parallel b-strands connected by 8 a-helical loops
– Active site is at bottom of funnel-shaped pocket
formed by loops
– Found in all glycolytic enzymes
– Found in proteins that bind and transport metabolites
 Biochemical question
Explain all of the following questions below:
 Structure of nucleoside and nucleotide.

 Purine nucleotide synthesis.

 Regulation control of Purine nucleotide synthesis.

 Purine Catabolism and Salvage pathway.

 Uric acid synthesis.

 Pyrimidine Ribonucleotide Synthesis.

 Regulatory Control of Pyrimidine Synthesis.

 Degradation of Pyrimidines.

 Abnormalities related to nucleotide metabolism