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Nucleic acid Chemistry

Dolores V. Viliran, M.D.


FEU-NRMF, Institute of Medicine
Department of Biochemistry
Function of Nucleic Acids
• Nucleic acid= an informational molecule
– Two kinds:
• DNA- deoxyribonucleic acid
• RNA- ribonucleic acid
– DNA = blueprint of living organisms
• Contains genetic information for the synthesis of
proteins
– RNA= machinery for the expression of the
coded information in DNA to proteins
• mRNA- Carrier of genetic information encoded in
the DNA
Two Kinds of Nucleic acid
• 1. DNA = deoxyribonucleic acid
• 2. RNA = ribonucleic acid
Primary function of DNA:Storage of genetic material

Central Dogma of LIFE : flow of genetic material


DNA  mRNA  protein

Replication  transcription  translation


PRESENT CONCEPT OF THE
CENTRAL DOGMA
DNA  RNA  PROTEIN

= specific transfer of information from RNA


to RNA has been observed in viral
systems
= specific transfer of information from RNA
to DNA has been observed in viral
systems and tumor.
How does DNA and RNA perform
these various functions?
• Structure is intimately related to function
• Change the structure and the function may
be lost
• Thus we need to know the structure in
order to understand the function
Covalent Structure of Nucleic acids
Genetic information is encoded in DNA and carried by
mRNA as sequence or a chain of nucleotides
• Composed of polymers of nucleoside
monophosphates(nucleotide-nucleotide-nucleotide)
1. Monomeric units consist of: NUCLEOTIDE
Base-sugar-phosphate (bsp)
a. Nitrogenous base which may be a purine or a pyrimidine
b. A pentose sugar, ribose or deoxyribose, in a furanose ring
form
c. A phosphate group esterified to the sugar

Nucleoside- coupling of a base and a sugar


Nucleotide- when a nucleoside becomes phosphorylated
• Nitrogenous base
– Purines = adenine(A), Guanine (G)
– Pyrimidines = cytosine (C),uracil (U), Thymine(T)
• Sugar
– DNA = deoxyribose
– RNA = ribose
• Sugar hold the base on one side and the
phosphate on the other side.
• Thus , sugar hold the components of the
nucleotide together
• Nucleotides in DNA
1) ADENINE-2-deoxyRIBOSE-PHOSPHATE
2) GUANINE-2-deoxyRIBOSE-PHOSPHATE
3) CYTOSINE-2-deoxyRIBOSE-PHOSPHATE
4) THYMINE-2-deoxyRIBOSE-PHOSPHATE
• Nucleotides in RNA
1) ADENINE-RIBOSE-PHOSPHATE
2) GUANINE-RIBOSE-PHOSPHATE
3) CYTOSINE-RIBOSE-PHOSPHATE
4) URACIL-RIBOSE-PHOSPHATE
• Nucleotides in DNA
– ADENINE-2-deoxyRIBOSE-PHOSPHATE
– GUANINE-2-deoxyRIBOSE-PHOSPHATE
– CYTOSINE-2-deoxyRIBOSE-PHOSPHATE
– THYMINE-2-deoxyRIBOSE-PHOSPHATE
• Nucleotides in RNA
– ADENINE-RIBOSE-PHOSPHATE
– GUANINE-RIBOSE-PHOSPHATE
– CYTOSINE-RIBOSE-PHOSPHATE
– URACIL-RIBOSE-PHOSPHATE
Chemical structure of nitrogenous bases

PURINES
Chemical structure of nitrogenous bases

PYRIMIDINES
SUBSTITUTIONS
OXYGEN,AMINO GROUPS,METHYL GROUPS
• PURINES
– ADENINE = 6-aminopurine
– GUANINE = 2-amino,6-oxypurine
• PYRIMIDINES
– CYTOSINE = 2-oxy,4-amino pyrimidine
– URACIL = 2,4-dioxypyrimidine
– THYMINE = 2,4-dioxy,5-methylpyrimidine
Nucleosides
Purines = N9-C1 glycosidic bond
Pyrimidines = N1-C1 glycosidic bond

• Addition of pentose sugar to a base


produces a nucleoside.
Adenine Adenosine
Guanine Guanosine
Cytosine Cytidine
Thymine Thymidine
Uracil Uridine
NUCLEOSIDE
NUCLEOTIDES
• Nucleotides can have one,two or three
phosphates
Nucleoside Nucleotide
Adenosine Adenosine mono/di/tri phosphate
Guanosine Guanosine mono/di/tri phosphate
Cytidine Cytidine mono/di/tri phosphate
Uridine Uridine mono/di/tri phosphate
Thymidine Thymidine mono/di/tri phosphate
AMP/ADP/ATP;GMP/GDP/GTP;CMP/CDP/CTP;
UMP/UDP/UTP;TMP/TDP/TTP
Covalent Structure of Nucleic acids
A. Nucleotides- are linked together by
phosphodiester bonds between the 3’ hydroxyl
on the sugar of one nucleotide and the 5’-
phosphate on the sugar of another nucleotide
1. Trinucleotides- addition of another nucleotide at the
3’ hydroxyl of the sugar
2. These compounds have polarity of direction- 5’ – 3’
3. Characteristically nucleotides are acidic by virtue of
their phosphate groups
NUCLEOTIDE POLYMERS
(NUCLEIC ACID)
Nomenclature of NUCLEIC ACIDS

A. Polynucleotides
B. Polyribonucleotides – polynucleotides containing
ribose as opposed to de-oxyribose
3. Naturally occuring are called RNA
4. Bases found in RNA’s (w/ a few exception) guanine,
cytosine, adenine, and uracil
C. Differences between the Primary Structure of DNA and
RNA
6. DNA has a 2’-deoxyribose as its sugar moiety rather
than ribose
7. DNA has one different base-thymine (5-methyl-uracil)
8. RNA has one different base-uracil
Ways of Presenting the structure of
a Polynucleotide (abbreviated form)
A. 5’ p-T-A-C-G-G-G-C-G-A-T-T-T-G-G-GOH³’
B. 5’ pTpApCpGpGpGpCpGpApTpTpTpGpGOH³
DNA – THE GENETIC MATERIAL
A. BASIC STRUCTURAL CHARACTERISTICS:
A double Helical Structure as proposed by Watson and
Crick (1953)
3. TWO ANTI – PARALLEL POLYDEOXY RIBONUCLEOTIDE
CHAINS – Wound around each other with the bases inside of the
helix sugar and PO4 – outside
4. STACKING OF THE HYDROHOBIC RINGS – Result when the
planes of the bases are perpendicular to the axis of the helix
5. HYDROGEN Bonding – between one bases on one stand pairing
w/ another base on the anti-parallel strand
Chargaff’s rule: content of A = content of T
content of G = content of C
4. The helix is 2 nm in diameter
= bases are separated by 3.4 A° along the helix
axis, and each base is rotated 36° in relation to
the previous base.
=the helical structure repeats at intervals of 34 A°
(every 10 base – pairs)
=In solution, the helical structure of DNA probably
does not repeat every 10-base pairs but nearer
to every 11 with the bases inclined 20° from the
horizontal
GENETIC INFORMATION IS STORED AS THE
SEQUENCE OF BASES ALONG THE CHAIN
Secondary structure of DNA: The
Double Helix
Right- and left-handed Helices
A, B, Z forms of DNA
Tertiary Structure of DNA
Supercoiling
• Supercoiling in prokaryotic DNA
– Positive supercoils
– Negative supercoils
• Supercoiling in Eukaryotic DNA
– Formation of chromatin
• electrostatic attraction between the negatively
charged phosphate groups on the DNA and the
positively charged groups on the protein
Supercoiling in prokaryotic DNA
Supercoiling in Eukaryotic DNA
Denaturation of DNA
• The double helix is disrupted during DNA
replication,transcription,repair and
recombination
• Therefore the forces that hold the two
strands together are adequate for
providing stability and yet weak enough to
allow easy strand separation
DISSOCIATION AND REASSOCIATION OF THE
DOUBLE HELICAL CHAINS OF DNA

Double helical chains of DNA have a remarkable ability to


dissociate from one another and to reassociate again.
This behavior is essential to the processes of
REPLICATION and TRANSCRIPTION
• DENATURATION – rupture of the hydrogen bonds
between the bases, resulting from increasing temperature
or the alteration of the H+ concentration, which causes the
two strands to come apart.
3. Increasing pH – deprotonates ring nitrogens of guanine
and thymine; decreasing pH protonates ring nitrogens of
adenine and cytosine
4. Increasing acidity – can cause rupture of purine glycosidic
bonds, and at high temperatures phosphodiester bonds
may be broken.
Alkali – method of choice for DNA denaturation
3. Increasing temperature – the double helical
chain dissociates at a definite temperature
known as Melting temperature (Tm)
c. Tm is the temp. at which 50% of the double
helix is unwound
d. Melting causes “unstacking of the base pairs”
which results in increased absorbance
(hyperchromic effect)
e. Tm is strongly influenced by the base
composition of the DNA
• DNA rich GC pairs has a higher Tm than
DNA with a higher proportion of AT pairs
• Mammalian DNA, which is about 40%
GC pairs has a Tm of about 87oC
B. RENATURATION (Annealing)
• If the temperature of melted
(dissociated)duplex DNA is rapidly
reduced, the original double helical
structure does not reform (anneal)
• If, however, the temp. is held at a value
of about 20oC to 25oC below the Tm, the
original double helical structure reforms.
Denaturation-Renaturation
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Types of DNA structure
1. Size of DNA is highly variable
a. DNA size can be expressed as number of
base pairs, molecular mass, the length of the
strands and actual mass of DNA
– DNA of molecular weight 1 x 106
contains 1500 bp and is 0.5 nm long
b. The amount of DNA per cell increases as the
complexity of the cellular function increases
- the DNA in the cell is packaged as 46
chromatin fibers
* metaphase- condensed state the largest chromosome
is about 10µm if stretched out is about 8 cm long
Types of DNA structure
1. Size of DNA is highly variable
2. Techniques of determining DNA size
a. Equilibrium centrifugation:Cesium
Chloride
b. Electron microscopy
c. Electrophoresis
Types of DNA structure
1. Size of DNA is highly variable
2. Techniques of determining DNA size
3. DNA maybe linear or circular
a. Double-stranded circles
b. single-stranded DNA
c. Circular DNA is a superhelix
SHAPE OF DNA MOLECULES
2. Chromosomal DNA may be linear or circular.
3. DNA is complexed with histones in the
eukaryotic chromosome, which profoundly
influence the structure of the chromosome.
4. Supercoiling-essential for stage of replication,
transcription and recombination it occurs only
when there is restraint upon the rotation of the
ends of the DNA chains, such as occurs in
circular DNA or in linear DNA when the ends
are to far apart to allow rapid spinning
Types of DNA structure
Alternative DNA conformations
a. DNA bending = DNA sequences with
runs of 4 to 6 Adenine bases phased by
10-bp spacers produce bend
conformations
= important in the interaction between
DNA sequences and proteins that
catalyze replication, transcription and
site-specific recombination
Types of DNA structure
Alternative DNA conformations
Cruciform DNA
= dos DNA (defined ordered sequence DNA)
- inverted repeat ( palindromes)
-mirror repeats
- Direct repeats
= present within noncoding DNA regions
= inverted repeats may function as a molecular switches
for replication and transcription
Triple-Stranded DNA
• Formed in DNA regions with
continuous string of of purine
bases, that is homopurine-
homopyrimidine regions
• Generated by the hydrogen
bonding of a third strand into
the major groove of B-DNA
• The third strand forms
hydrogen bonds with another
surface of the double helix
thru Hoogsteen pairs
• Limited to only four triplet
bases –TAT,CGC,GGC,AAT
Triple-stranded DNA
• Polypurine-polypyrimidine
regions have potential
biological roles: transcription
control,initiation of
replication, replication
terminators,enhancers of
stability of
telomeres,initiators of genetic
recombination
Four-Stranded DNA
(Quadruplex)
• May form during DNA
recombination
• Contains repeated
motifs high in guanine
content (G-quartet
DNA)
• Form at telomeres
Slipped DNA
• Slipped,mispaired DNA
(SMP-DNA)
• Formation involves the
unwinding of the double
helix and realignment and
subsequent pairing of one
copy of the direct repeat
with an adjacent copy on
the other strand-
generating a single-
stranded loop
EUKARYOTIC DNA:
1. DNA is packaged into unit structures called
CHROMOSOMES
2. Combined with proteins called CHROMATIN chromatin
contains about equal amounts (by weight) of DNA and
protein.
3. DNA is associated with basic proteins called histones
and with nonhistones chromosomal proteins. These
are non-covalent associations
d. Histones are small proteins that carry a considerable
(+) charge devided into 5 classes H1;H2A;H2B, H3
and H4
Chromosomal Organization
• If the DNA of all 46 chromosomes were
lined up in the B-DNA conformation it
would be more than 2 meters long
• DNA needs to fit within a cell nucleus with
a diameter of approximately 5 µm
• Packaging of DNA
-Beads-on-a-string: DNA association with histones
- 10 nm fiber
- 30 nm fiber (solenoid)
Histone Octamer
inserted into the nucleosome
CYTOPLASMIC DNA IN
EUKARYOTIC ORGANELLES:
Mitochondria and chloroplasts of eukaryotic cells contain
DNA that differs from nuclear DNA
2. Neither mitochondrial nor chloroplasts DNA is
associated with histones
3. Mitochondrial DNA of animal cells is:
d. Double – stranded
e. Circular
f. About 15,000 base-pairs in length
3. Mitochondrial Dna sequences code for only about 5% of
the protein components of mitochondrial structure and
function. The bulk of information for mitochondrial
protein synthesis is stored in nuclear DNA
CHEMICAL NATURE OF RNA;
1. Sugar moiety is ribose rather than 2’
deoxyribose of DNA
2. Pyrimidine component is URACIL
ribonucleotide
3. Exists as a single strand

mRNA
Cap Poly-A tail

5’Gppp A,C,G,U,G,A,U,G,A,C,U,U,A,G,G,C, pApApApApApA-OH 3’


mRNA
Characteristics:
2. Most frequently synthesized and with
most rapid turnover among the RNA’s
3. Synthesized in transcription
4. Carries the genetic information from the
DNA (codons- triplet nucleotide) and is
used as the template for protein
synthesis
5. Single stranded
6. Read and translated 5’ to 3’
7. Has methylguanosine cap at the 5’ end
and a polyadenine tail
tRNA
Characteristics:
3. With the lowest molecular weight
4. Synthesized during transcription
5. With about 60 different types, with atleast one tRNA for every AA
some amino acids have 2 or more corresponding tRNA’s
6. Transfers amino acids from the cytoplasm to the ribosomes
7. “adaptor” molecule that carries specific amino acid to the site of
protein synthesis. There, if recognizes the genetic code word that
specifies the addition of its amino acid to the growing peptide chain
8. Contains the anti-codon
9. Single stranded but assumes a cloverleaf structure (secondary level)
10. Contains unusual/rare bases like ribothymine, pseudouracil and
dihydroxyuracil
11. Amino acid is always attached to the 3’ end and the 3’ end always
end in CCA sequence
12. Amino acid is attached to the tRNA during the activation stage of
translation
tRNA
rRNA
Characteristics:
3. Intimately associated with ribosomes
4. Most abundant of all RNA’s
5. Least characterized among all RNA’s
and is synthesized during transcription
rRNA
METABOLISM OF PURINE AND
PYRIMIDINE NUCLEOTIDES
Biomedical Importance
The biosynthesis of purines and pyrimidines is
stringently regulated and coordinated by
feedback mechanisms that ensure their
production in quantities and at times appropriate
to varying physiologic demand
Genetic diseases of purine metabolism include
gout, Lesch-Nyhan syndrome, adenosine
deaminase deficiency, and purine nucleoside
phosphorylase deficiency
By contrast, apart from the orotic acidurias, there
are few clinically significant disorders of
pyrimidine catabolism.
Purines & Pyrimidines Are Dietarily Nonessential
• Human tissues can synthesize purines and pyrimidines from
amphibolic intermediates
• Ingested nucleic acids and nucleotides, which therefore are
dietarily nonessential, are degraded in the intestinal tract to
mononucleotides, which may be absorbed or converted to
purine and pyrimidine bases
• The purine bases are then oxidized to uric acid, which may be
absorbed and excreted in the urine
• While little or no dietary purine or pyrimidine is incorporated
into tissue nucleic acids, injected compounds are incorporated
• The incorporation of injected [3H]thymidine into newly
synthesized DNA thus can be used to measure the rate of
DNA synthesis.
Role of Nucleotides in Biochemical Processes:
1. Activated precursors of DNA and RNA
2. Activated intermediates in many biosynthetic reactions
C. UDP GLUCOSE – Glycogen synthesis
D. CDP GLYCEROL – Phosphoglyceride synthesis
E. s-ADENOSYL METHIONINE – methylation reactions
3. Energy Sources
G. ATP – universal energy currency
H. GTP – translocation of nascent peptide chains on
ribosomes
- Activation of signal coupling proteins
4. ADENINE NUCLEOTIDES – COMPONENTS OF THREE
CO-ENZYMES: NAD, FAD, AND Co-A
5. METABOLIC REGULATORS
i.e. c-AMP – intra-cellular secondary messenger
Biosynthesis of Purine Nucleotides
Purine Nucleotide Biosynthesis
• Three processes contribute to purine
nucleotide biosynthesis
(1) synthesis from amphibolic intermediates
(synthesis de novo)
(2) phosphoribosylation of purines
(3) phosphorylation of purine nucleosides.
Inosine Monophosphate (IMP) Is
Synthesized from Amphibolic Intermediates
Conversion of IMP to AMP and GMP
"Salvage Reactions" Convert Purines &
Their Nucleosides to Mononucleotides
Reduction of Ribonucleoside Diphosphates
Forms Deoxyribonucleoside Diphosphates
Regulation of the reduction of purine and
pyrimidine ribonucleotides to their respective
2'-deoxyribonucleotides
Biosynthesis of Pyrimidine Nucleotides
PURINE NUCLEUS
SOURCES:
• N-3 and N-9 from the amide nitrogen of Gln
• C-4, C-5 and N7 from glycine
• C-2 form N10 formyl H4 folate
C-8 from N5 N10 methenyl H4 folate
4. C-6 from CO2
5. N-1 from aspartate
Pyrimidine Nucleus
SOURCES:
10. N-1, C-4, C-5 and C-6 from Asp
11. C2 from CO2
12. N3 from the amide N of Gln (glutamine)
FORMATION OF DEOXYRIBONUCLEOTIDES
ADP dADP
GDP dGDP
UDP dUDP
CDP dCDP
-produces dNTPs for DNA replication
-enhanced during the S phase of cell cycle (prior to cell
division)
-formed from the reduction of the corresponding
ribonucleosides diphosphates.
-enzyme ribonucleoside reductase
direct reducing of Hydrogen donor: thioredoxin (Sh
containing protein)
ultimate hydrogen donor: NADPH
enzyme to re-reduce thioredoxin: thioredoxin reductase
-subject to regulation
FORMATION OF DEOXYRIBONUCLEOTIDES

NMP
ATP
ADP
NDP
Ribonucleotide thioredoxin (SH)2 NADP
Reductase
thioredoxin (S-S) NASPH+H
dNDP

ATP

ADP

dNTP
FORMATION OF TMP
-In the formation of dTMP from dUMP, FH4 is the methyl donor which
simultaneously is oxidized to FH2, this requiring reduction by dihy
drofolate reductase.
-This reaction is most sensitive to the inhibitory effect of aminopterin or
amethopterin (methotrexate).
-Aminopterin is an analogue of folic acid thus competitively inhibiting
dihydrofolate reductase, preventing the formation of FH4, the active
form of the enzyme.
Hence, this drug is used in cancer chemotherapy because of TMP
synthesis, and ultimately DNA replication.
-Trimethoprim (present in Bactrim, Cotrimoxazole) has similar
mechanism of action but acts more on bacterial cells, hence used
as antibiotics.
-Trimethoprim is generally used in combination with sulfonamides, a
para-aminobenzonic acid (PABA) analogue. PABA is a constituents
of folic acid, hence the presence of sulfonamides will prevent folic
acid synthesis in bacteria (but not in man, as folic acid is ingested
pre-formed in man as a vitamin), thus inhibiting TMP synthesis and
DNA replication of bacterial leading to cell death
FORMATION OF TMP
dUTP dCPM
Hydrolase Deaminase
Ppi NH3

dUMP
N5 N10 methylene
FH4
Dihydrofolate Thymidylate
Reductase synthetase

FH2 dMTP
SALVAGE PATHWAYS FOR PURINE AND
PYRIMIDINE BASES
-important in cells which cannot carry out the de
novo pathways e.g. due to lack of enzyme PRPP
amidotransferase (rate limiting step in purine
nucleotide biosynthesis) as in red blood cells.
-more economical in terms of high energy
phosphates expenditure compared to the de
novo pathway.
-nucleosides, which can enter cells (nucleotides
cannot) are either converted to a nucleotide thru
the action of a kinase or degraded to a base by
nucleoside phosphorylase
ENZYMES
1. Hypoxanthine – guanine phosphoribosyl
transferase (HGPRTase) catalyzes the one-
step formation of hypoxanthine or guanine to
IMP or GMP respectively
Guanine + PRPP GMP+PPi
Hypoxanthine + PRPP IMP + PPi
Complete deficiency of this enzyme produces
Lesch-Nyhan Syndrome characterized by:
1. Hyperuricemia and gout
2. Psychomotor abnormalities
3. Mental retardation
4. Self mutilation
2. Adenine phosphoribosyl transferase (APRTase)
catalyzes the conversion of adenine to AMP
Adenine + PRPP AMP+PPi
3. Pyrimidine phosphoribosyl transferase catalyzes
the conversion of orotate, uracil and thymine to
their corresponding nucleotides.
Orotate+PRPP OMP+PPi
Uracil + PRPP UMP+PPi
Thymine + PRPP TMP+PPi
FORMATION OF TMP
-In the formation of dTMP from dUMP, FH4
is the methyl donor which simulatneously
is oxidized to FH2, this requiring reduction by
dihy drofolate reductase.
-This reaction is most sensitive to the inhibitory
effect of aminopterin or amethopterin
(methotrexate)
FORMATION OR DEOXYPYRIMIDINE
NUCLEOTIDES FOR DNA SYNTHESIS

CDP dCDP dCMP dUMp

dCTP dTMP

DNA dTDP

dTTP
SALVAGE PATHWAYS
FREE BASE (PURINES, PYRIMIDINES)
RIBOSE-1-P
PRPP PI
PPi nucleosides

NMP ATP
ATP Kinase
NDP
ATP Kinase

NTP
Degradation of Pyrimidine Bases:
Deaminase reductase
Cytosine Uracil Dihydrouracil
H20 NH4+ NADPH NADP
H20
B-Ureidopropionate

B-alanine

CO2+NH3

B-aminoisobutyrate
B-ureidoisobutyrate

deaminase reductase H20


Methyl cytosine thymine dihydrothymine
H20 NH4 NADPH NADP
-Ring opening gives rise to B-
ureidopropionate and B-ureidoisobutyrate
-uracil and cytosine eventually gives rise to
B-alanine
-B-aminosobutyrate, the degradation
product of thymine can be used to
measure DNA turnover
Degradation of Pyrimidine Bases:
Deaminase reductase
Cytosine Uracil Dihydrouracil
H20 NH4+ NADPH NADP
H20
B-Ureidopropionate

B-alanine

CO2+NH3

B-aminoisobutyrate
B-ureidoisobutyrate

deaminase reductase H20


Methyl cytosine thymine dihydrothymine
H20 NH4 NADPH NADP
Disorders of Purine Catabolism
HYPERURICEMIA
1. Gout
2. Lesch-Nyhan syndrome
defect in hypoxanthine-guanine phosphoribosyl
transferase
3. Von Gierke's Disease
hyperuricemia in von Gierke's disease (glucose-6-
phosphatase deficiency) occurs secondary to
enhanced generation of the PRPP precursor ribose 5-
phosphate
An associated lactic acidosis elevates the renal
threshold for urate, elevating total body urates.
Disorders of Purine Catabolism
HYPOURICEMIA
Hypouricemia and increased excretion of hypoxanthine and xanthine
1. xanthine oxidase deficiency due to a genetic defect or to severe liver
damage. Patients with a severe enzyme deficiency may exhibit
xanthinuria and xanthine lithiasis.
2. Adenosine Deaminase & Purine Nucleoside Phosphorylase Deficiency
Adenosine deaminase deficiency is associated with an immunodeficiency
disease in which both thymus-derived lymphocytes (T cells) and bone
marrow-derived lymphocytes (B cells) are sparse and dysfunctional
Purine nucleoside phosphorylase deficiency is associated with a severe
deficiency of T cells but apparently normal B cell function
Immune dysfunctions appear to result from accumulation of dGTP and
dATP, which inhibit ribonucleotide reductase and thereby deplete cells of
DNA precursors.
Diagnosis:
-Colchicine trial
-synovial fluid finding
• needle shaped
• Negatively birefringent
• Wbc count 10,000 ~ 60,000
• Neutrophil predominance
-Tophi
• Cream colored, firm, chalky, movable
• Punched-out erosions
• Overhanging edge
Treatment
-colchicine
-NSAIDs(indomethacin)
-Corticosteroids
-allopurinol (xanthine oxidase inhibitor)
-Probenecid (uricosuric)
Diet therapy:
Purines: low
Calories: maintain ideal weight
Carbohydrates: high
Proteins: moderate
Fats: low
Fluids: large amounts
Foods:
High Purine Content
Anchovies Bouillon Brain Broth
Goose Gravy Heart Herring
Kidney Liver Mackerel yeast
Meat extracts & mince mussels portridge
Roe sardines scallops sweetbread
Moderate purine content
Meat & fish except those listed above
Poultry shellfish asparagus beans
Lentils mushroom peas spinach

Negligible purine content


Bread crackers butter margarines
Cake cereal cheese chocolate
Coffee cornbread ice cream milk
Noodles nuts oils olives
Pickles popcorn pudding rice
Soft drinks eggs fruits gelatin
Tea vegetables (except those listed above)
Disease associated with defects of
nucleotide metabolism
1. Hyperuricemia and gout
Deposition of tophi in soft tissues and joints
Can be due to inability to regulate the production of purine nucleotides e.g.
overactivity of PRPP synthetase or PRPP amidotransferase
2. Lesch-Nyhan Syndrome
Defeciency of HGPRT ase (enzyme of the salvage pathway)
3. Von-Gierke’s disease- the deficiency of glucose-6-phosphatase
increases the levels of glucose-6-P which in turn enhance the
pentose phosphate pathway (HMP shunt) increasing intracellular
levels of ribose -1 phosphate. Elevated levels of ribose-1-phosphate
increases cellular production of PRPP with consequent
overproduction of purine nucleotides, thus HYPERURICEMIA
4. Hypouricemia associated with deficiency of xanthine oxidase or purine
nucleoside phosphorylase (no degredation of purines to uric acid)
can be due to severe liver damage. Can exhibit xanthinuria and
xanthine lithiasis if deficiency is severe.
5. Immunodeficiency diseases associated with
adenosine deaminase deficiency
-there is dysfunction of T-cells and B-cells
apparently due to the inhibitory effect of dATP
which accumulates in this disorder dATP is a
strong inhibitor of this ribonucleotide reductase,
an important enzyme in the synthesis of dNTPs
depleting cells of DNA precursors particularly
dCTP.
6. Immunodeficiency diseases associated with
purine nucleoside phosphorylase deficiency.
-impaired T-cell function but normal B-cell
Catabolism of Pyrimidines Produces
Water-Soluble Metabolites
Catabolism of Pyrimidines
• the end products of pyrimidine catabolism
are highly water-soluble: CO2, NH3,
-alanine, and –aminoisobutyrate
• Excretion of -aminoisobutyrate increases
in leukemia and severe x-ray radiation
exposure due to increased destruction of
DNA.
Disorders associated with defects
in pyrimidine metabolism
1. Orotic aciduria
-characterized by high levels of urinary orotic acid, retarded
growth and megaloblastic anemia.
-there is a deficiency of orotate phosphoribosyl transferase,
orotate decarboxylase or both which prevents the
formation of UTP or CTP – inhibiting the carbamoyl
phosphate synthetase step or ATCase step
respectively.
-the failure to synthesize pyrimidine nucleotides is also the
cause for growth retardation and megaloblastic
anemia.
-can be treated by giving uridine (make pyrimidine essential
constituents of the diet since the de novo pathway
cannot provide adequate amounts of pyrimidine
nucleotide synthesis. This will also exert a feedback
inhibition on carbamoyl phosphate synthetase reaction
2. Ornithine Transcarbamoylase Deficiency (OTCD)
-Characterized by orotic aciduria
-due to accumulation of carbamoyl phosphate
-deficiency of this enzyme prevents the formation of
citrulline from the reaction between carbamoyl
phosphate and ornithine (part of the urea cycle) hence
producing elevated levels of carbamoyl phosphate.
-elevated levels of carbamoyl phosphate in turn stimulates
the rate limiting step in the pyrimidine synthesis pathway
producing elevated levels of orotic acid
-manifestation of protein intolerance and hyperammonemia
with hepatic encephalophaty are also apparent because
of the failure to detoxify ammonia as a consequence of
the deficiency of the enzyme.
INHIBITORS OF PURINE METABOLISM
(FOR CANCER THERAPY)
The turnover of nucleic acids in malignant tissues is very
high, thus inhibition in its formation may have a
therapeutic effect. Can also affect normal host cells
which are rapidly dividing like the hematopoietic
system.
2. 6-mercaptopurine (6MP)-an anti-tumor drug: an
analogue of adenine metabolized to a ribonucleotide by
the APRT salvage pathway thus inhibiting conversion
of IMP to GMP or AMP. It also inhibits the rate-limiting
step of the de novo pathway. Simultaneous
administration with allopurinol potentiates its effects as
6MP eill not be degraded and therefore will delay its
activation.
2. Adenosine arabinoside (sugar is replaced
by arabinose)- used as an antiviral and
anti-tumor agent in man; inhibits DNA
polymerase after it has been metabolized
to the triphosphate form.
3. Azaserine-an analogue of Gln, thus,
inhibits the incorporation of N9 and N3 into
the purine ring, inhibits formation of GMP
from IMP, CTP from UTP – reactions
requiring GLN.
Inhibitors to pyrimidine metabolism
for cancer/viral chemotherapy
1. Aminopterin and methotrexate – inhibits dihydrofolate
reductase
2. 5-flouduroucil (5-FU)
-an analogue of thymine used in the treatment of solid
tumors converted to the monophosphate nucleotide
form via the salvage pathway
-eventually converted to the deoxyribonucleotide form and
binds to thymidylate synthetase, thereby inhibiting the
formation of TMP.
-as a deoxytriphosphate form, can be incorporated into
RNA and inhibits the formation of mature RNA
(important in translation)
3. 5-Iodouracil
-functions as an analogue of thymidine
when incorporated into DNA, bonds with C
rather that A, thus causing a misreading in
DNA sequence
4. Cytosine arabinoside
-used in the treatment of leukemias has a
very short half-life
-inhibits DNA polymerase in its triphosphate
form
Summary
• Ingested nucleic acids are degraded to purines and
pyrimidines. New purines and pyrimidines are formed
from amphibolic intermediates and thus are dietarily
nonessential.
• Several reactions of IMP biosynthesis require folate
derivatives and glutamine. Consequently, antifolate
drugs and glutamine analogs inhibit purine biosynthesis.
• Oxidation and amination of IMP forms AMP and GMP,
and subsequent phosphoryl transfer from ATP forms
ADP and GDP. Further phosphoryl transfer from ATP to
GDP forms GTP. ADP is converted to ATP by oxidative
phosphorylation. Reduction of NDPs forms dNDPs.
Summary
• Hepatic purine nucleotide biosynthesis is stringently
regulated by the pool size of PRPP and by feedback
inhibition of PRPP-glutamyl amidotransferase by AMP
and GMP.
• Coordinated regulation of purine and pyrimidine
nucleotide biosynthesis ensures their presence in
proportions appropriate for nucleic acid biosynthesis and
other metabolic needs.
Summary
• Humans catabolize purines to uric acid (pKa 5.8),
present as the relatively insoluble acid at acidic pH or
as its more soluble sodium urate salt at a pH near
neutrality. Urate crystals are diagnostic of gout. Other
disorders of purine catabolism include Lesch-Nyhan
syndrome, von Gierke's disease, and hypouricemias.
• Since pyrimidine catabolites are water-soluble, their
overproduction does not result in clinical
abnormalities. Excretion of pyrimidine precursors can,
however, result from a deficiency of ornithine
transcarbamoylase because excess carbamoyl
phosphate is available for pyrimidine biosynthesis.
This DNA form is seen physiologic conditions
where cell is well hydrated:

• A. A form
• B. B form
• C. Z form
• D. D form
This DNA form is seen physiologic conditions
where cell is well hydrated:

• A. A form
• B. B form
• C. Z form
• D. D form
TRUE regarding salvage pathways of
nucleotide metabolism:
• A. They utilize free bases as substrates for
nucleotide biosynthesis
• B. They take place only in the mitochondria
• C. They derive the ring from amphibolic
intermediates
• D. They are more efficient than de novo pathway
TRUE regarding salvage pathways of
nucleotide metabolism:
• A. They utilize free bases as substrates for
nucleotide biosynthesis
• B. They take place only in the mitochondria
• C. They derive the ring from amphibolic
intermediates
• D. They are more efficient than de novo pathway
The enzyme that catalyzes the committed
step in purine de novo pathway:

• A. Xanthine oxidase
• B. PRPP synthase
• C. PRPP glutamyl amido transferase
• D. HGPRTase
The enzyme that catalyzes the committed
step in purine de novo pathway:

• A. Xanthine oxidase
• B. PRPP synthase
• C. PRPP glutamyl amido transferase
• D. HGPRTase
Xanthine Oxidase inhibitor
• A. Azaserine
• B. Sulfonamides
C. Trimethoprim
• D. allopurinol
Xanthine Oxidase inhibitor
• A. Azaserine
• B. Sulfonamides
C. Trimethoprim
• D. allopurinol
The first purine nucleotide synthesized
during the de novo pathway

• A. AMP
• B. GMP
• C. IMP
• D. ADP
The first purine nucleotide synthesized
during the de novo pathway

• A. AMP
• B. GMP
• C. IMP
• D. ADP
Which enzyme participates in both salvage
and De novo pathway?

• A. Adenosine phosphoribosyl transferase


• B. Xanthine oxidase
• C. Phosphoribosyl phosphate synthase
• D. Phosphoribosyl phosphate
amidotransferase
Which enzyme participates in both salvage
and De novo pathway?

• A. Adenosine phosphoribosyl transferase


• B. Xanthine oxidase
• C. Phosphoribosyl phosphate synthase
• D. Phosphoribosyl phosphate
amidotransferase
The primary product of purine
catabolism
• A. Xanthine
• B. Urea
• C. Uric acid
• D. hypoxanthine
The primary product of purine
catabolism
• A. Xanthine
• B. Urea
• C. Uric acid
• D. hypoxanthine
Which molecule is not a
monomer of ribonucleic acids?
• A. Adenine
• B. Thymine
• C. Cytidine
• D. Uridine
Which molecule is not a
monomer of ribonucleic acids?
• A. Adenine
• B. Thymine
• C. Cytidine
• D. Uridine
Storage molecule of genetic
information:
• A. DNA
• B. RNA
• C. Both
• D. Neither
Storage molecule of genetic
information:
• A. DNA
• B. RNA
• C. Both
• D. Neither
Hyperchromicity effect is observed in:

• A.DNA renaturation
• B. DNA denaturation
• C. dsDNA
• D. RNA
Hyperchromicity effect is observed in:

• A.DNA renaturation
• B. DNA denaturation
• C. dsDNA
• D. RNA
If the adenine content of a double-helical
DNA is 20% of the total bases, the cytosine
content would be:

A. 20%
B. 30%
C. 40%
D. 50%
E. 60%
If the adenine content of a double-helical
DNA is 20% of the total bases, the cytosine
content would be:

• 20%
• 30%
• 40%
• 50%
E. 60%
END OF LECTURE