Transcript Nucleotides

UNIT IV:
Nitrogen Metabolism
Nucleotide Metabolism
1- Overview
 Nucleotides are essential for all cells
 DNA and RNA synthesis/protein synthesis/cell proliferation
 Carriers of activated intermediates in synthesis of some CHO’s,
lipids and proteins
 Structural components of several essential coenzymes, e.g., CoA,
FAD, NAD+, NADP+
 cAMP and cGMP serve as second messengers in signal transduction
 Energy currency
 Regulatory compounds for many pathways of intermediary
metabolism
 Purine and pyrimidine bases can be synthesized de novo, or obtained
through salvage pathways
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2- Nucleotide structure
 Nucleotides are composed of:
 a nitrogenous base (purine/pyrimidine),
 a pentose,
 and 1, 2 or 3 phosphate groups.
A. Purine and pyrimdine structures
 Both DNA and RNA contain purine bases: adenine (A) and
guanine (G).
 Both DNA and RNA contain cytosine (C)
 DNA contains thymine (T), whereas RNA contains uracil (U).
 T and U differ by one methyl group
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Unusual bases are occasionally
found in some species of DNA and
RNA
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e.g., in some viral DNA, tRNA
Base modifications include
methylation, hydroxymethylation,
glycosylation, acetylation, or
reduction
• May aid in recognition by specific
enzymes/proteins, or protect
from degradation by nucleases
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B. Nucleosides
 Pentose sugar + N-base = nucleoside (ribonucleoside/
deoxyribonucleosides).
 If sugar is ribose, ribonucleoside is produced
 Ribonucleosides of A, G, C and U = adenosine, guanosine,
cytidine, uridine
 If sugar is deoxyribose, a deoxyribonucleoside is
produced
 Deoxyribonucleosides have the added prefix “deoxy”
 eg. deoxyadenosine
 Carbon and nitrogen atoms in base and sugar are
numbered separately (Figure 22.3B)
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• Note that the carbons in the
pentose are numbered 1' to
5‘
• Thus, when the 5'-carbon of
a nucleoside (or nucleotide)
is referred to, a carbon
atom in the pentose, rather
than an atom in the base, is
being specified.
Figure 22.3
A. Pentoses found in nucleic acids.
B. Examples of the numbering
systems for purine- and pyrimidine
containing nucleosides.
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C- Nucleotides
 Nucleotides are mono-, di-, or triphosphate esters of nucleosides.
 The 1st phosphate group is attached by an ester linkage to the 5`
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OH of pentose.
This compound is called nucleoside 5`-phosphate or 5`nucleotide
If one phosphate is attached, the structure is a nucleoside
monophosphate (NMP) e.g., AMP, CMP
If a 2nd or 3rd phosphate is added, a nucleoside diphosphate (e.g.,
ADP) or triphosphate (e.g., ATP) (Figure 22.4)
The 2nd and 3rd phosphates are connected by a ”high-energy”
bond.
Phosphate groups give negative charges to nucleotides, and cause
DNA and RNA to be referred to as “nucleic acids”.
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3- Synthesis Of Purine Nucleotides
 Atoms of purine ring are
contributed by: Asp, Gly and Gln/
CO2/ and N10-formyltetrahydrofolate
 Ring is constructed in the liver by
reactions that add donated carbons
and nitrogens to preformed ribose
5-phosphate.
Figure 22.5
Sources of individual atoms in
the purine ring
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A. Synthesis of 5-phosphoribosyl-1pyrophosphate (PRPP)
 PRPP is an “activated pentose”, participates in synthesis
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of purines and pyrimidines, and in salvage of purine bases
 Synthesis of PRPP from ATP and ribose-5-P is catalyzed
by PRPP synthetase (ribose phosphate
pyrophosphokinase).
 The enzyme is activated by the inorganic phosphate (Pi)
and inhibited by purine nucleotides (end-product)
 Sugar in PRPP is ribose, ribonucleotides are the end
products of de novo purine synthesis
 When deoxyribonucleotides needed for DNA syhtnesis,
ribose is reduced
Figure 22.6
Synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP), showing the
activator and inhibitors of the reaction.
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B. Synthesis of 5`-phosphoribosylamine
 Amide group of Glutamine replaces the pyrophosphate
group attached to C1 of PRPP
 The enzyme, glutamine: phosphoribosyl pyrophosphate
amidotransferase, is inhibited by purine 5` nucleotides AMP,
GMP (end products).
 This is the committed step in purine nucleotide biosynthesis
 The rate of reaction is also controlled by Glutamine and
PRPP conc. (intracellular PRPP conc. is normally far below
the Km for the amidotransferase, i.e., small changes in
[PRPP] cause proportional change)
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C. Synthesis of inosine monophosphate,
the “parent” purine nucleotide
 The next 9 steps in purine nucleotide biosynthesis lead to
the synthesis of Inosine Monophosphate (IMP, whose base is
hypoxanthine).
 The pathway requires 4 ATPs.
 Two steps require N10-formyltetrahydrofolate.
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Figure 22.7
Synthesis of purine
nucleotides, showing the
inhibitory effect of some
structural analogs.
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D. Synthetic inhibitors of purine synthesis
 Sulfonamides, synthetic inhibitors, inhibit growth of
rapidly dividing microorganisms without interfering with
human cell functions.
 Other purine synthesis inhibitors (eg. Methotrexate,
structural analog of folic acid) used to control spread of
cancer by interfering with synthesis of nucleotides (thus
DNA, RNA).
 Trimethoprim (another folate analog) has antibacterial
activity as it selectively inhibits bacterial dihydrofolate
reductase.
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D. Synthetic inhibitors of purine synthesis
 Inhibitors of human purine synthesis are extremely toxic to
tissues, especially to developing structures such as in a fetus ,
or to cell types that replicate rapidly, including those of BM,
skin, GI tract, immune system, or hair follicles.
 Individuals taking such anti-cancer drugs experience adverse
effects e.g., anemia, scaly skin, GI tract disturbance,
immunodeficiencies, and baldness
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E. Conversion of IMP to AMP and GMP
 This requires a 2-step energy requiring pathway
 The synthesis of AMP requires guanosine triphosphate
(GTP) as an energy source, whereas the synthesis of GMP
requires ATP
 The 1st reaction in each pathway is inhibited by the end
product.
 This diverts IMP to the synthesis of the purine species
present in lesser amounts.
 If both AMP and GMP present in adequate amounts, de novo
pathway of purine synthesis is turned off at amidotransferase
step.
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Figure 22.8
Conversion of IMP to AMP and GMP showing feedback inhibition.
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F. Conversion of nucleoside monophosphates to
nucleoside diphosphates and triphosphates
 Nucleoside diphosphates (NDPs) are synthesized from Nucleoside
monophosphates (NMPs) by base-specific nucleoside monophosphate
kinases
 These kinases do not discriminate between ribose or deoxyribose in the
substrate.
 ATP is generally the source of transferred phosphate as it is the
abundant Nucleoside Triphosphate (NTP)
 Adenylate kinase is particularly active in liver and muscle, where
turnover of energy from ATP is high.
 Its function is to maintain an equilibrium among AMP, ADP and ATP
 NDPs and NTPs are interconverted by nucleoside diphosphate kinase,
an enzyme with broad specificity.
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Figure 22.9
Conversion of nucleoside monophosphates to nucleoside
diphosphates and triphosphates.
G. Salvage pathway of purines
 Purines from normal turnover of cellular nucleic acids, or
obtained from diet and not degraded, can be reconverted
into NTPs and used by the body
 This is referred to as the “salvage pathway” of purines.
1. Conversion of purine bases to nucleotides:
 Two enzymes are involved:
 Adenine phosphoribosyltransferase (APRT) and
 Hypoxanthine-guanine phosphoribosyltransferase (HPRT)
 Both enzymes use PRPP as source of ribose 5-p.
 The release of pyrophospahte (ppi) makes these reactions
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irreversible.
 Adenosine is the only purine nucleoside to be salvaged
.
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2. Lesch-Nyhan syndrome:
 X-linked, recessive disorder, associated with virtually complete
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deficiency of HPRT.
Inability to salvage hypoxanthine or guanine, from which excessive
amounts of uric acid are produced.
In addition, lack of salvage pathway causes increased PRPP levels and
decreased IMP and GMP levels.
So, glutamine:phosphoribosylpyrophosphate amidotransferase has excess
substrate and decreased inhibitors available, and de novo purine
synthesis increased.
Decreased purine reutilization and increased purine synthesis results in
production of large amounts of uric acid, making Lesch-Nyhan
syndrome a severe, heritable form of gout.
Patients with Lesch-Nyhan syndrome tend to produce urate kidney
stones.
In addition, characteristic neurologic features of the disorder include
self-mutilation (biting of lips and fingers) and involuntary movements.
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4. Synthesis of Deoxyribonucleotides
 The nucleotides describes thus far contain ribose
 The nucleotides required for DNA synthesis, however,
are 2'-deoxyribonucleotides, which are produced from
ribonucleoside diphosphates by the enzyme
ribonucleotide reductase
 The same enzyme acts on pyrimidine ribonucleotides.
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A- Ribonucleotide reductase
 It is a multi-subunit enzyme composed of two
nonidentical dimeric subunits, 2B1 and 2B2
 Specific for the reduction of nucleoside diphosphates
(ADP, GDP, CDP, UDP) to their deoxy forms.
 The immediate donors of hydrogen atoms needed for
reduction of 2`-OH are two –SH groups on the enzyme
itself, which during reaction form a disulfide bond.
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Figure 22.12
Conversion of
ribonucleotides to
deoxyribonucleotides.
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A- Ribonucleotide reductase
1. Regeneration of reduced enzyme.
 In order for ribonucleotide reductase to continue to produce
deoxyribonucleotides, the disulfide bond created during the
production of the 2'-deoxy carbon must be reduced
 The source of the reducing equivalents for this purpose is
thioredoxin—a peptide coenzyme of ribonucleotide reductase
 Thioredoxin contains two cysteine residues separated by two amino
acids in the peptide chain.
 The two –SH groups of thioredoxin donate their H atoms to the
enzyme, in the process forming S-S bond.
2. Regeneration of reduced thioredoxin.
 The necessary reducing equivalents are provided by NADPH & H+,
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and the reaction is catalyzed by thioredoxin reductase (see Figure
22.12)
B. Regulation of deoxyribonucleotide
synthesis
Ribonucleotide reductase is responsible for maintaining a
balanced supply of the deoxyribonucleotides required for DNA
synthesis.
 Regulation of the enzyme is complex.
 In addition to the single catalytic (active) site, there are
allosteric sites on the enzyme involved in regulating its activity.
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1. Activity sites:
 The binding of dATP to allosteric sites (known as the activity sites)
on the enzyme inhibits the overall catalytic activity of the enzyme
and therefore prevents reduction of any of the four NDPs.
 This effectively prevents DNA synthesis, and explains the toxicity of
increased levels of dATP seen in conditions such as adenosine
deaminase deficiency.
 In contrast, ATP bound to these sites activates the enzyme.
B. Regulation of deoxyribonucleotide
synthesis
2.
Substrate specificity sites:
 The binding of nucleoside triphosphates to additional allosteric
sites (known as the substrate specificity sites) on the enzyme
regulates substrate specificity, causing an increase in the
conversion of different species of ribonucleotides to
deoxyribonucleotides as they are required for DNA synthesis.
 E.g., deoxythymidine triphosphate (dTTP) binding at the
specificity sites causes a conformational change that allows
reduction of GDP to dGDP at the catalytic site.
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• The drug, hydroxyurea destroys the
free radical required for enzymatic
activity of ribonucleotide reductase,
and thus inhibits the generation of
substrates for DNA synthesis.
• Hydroxyurea has been used in the
treatment of cancers such as chronic
myelogenous leukemia.
• Hydroxyurea is also used in the
treatment of sickle cell disease
however, the increase in fetal
hemoglobin seen with hydroxyurea
has not been linked to its effect on
ribonucleotide reductase.
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