21' Deoxyribonucleotide biosynthesis and nucleotide catabolism

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21. Deoxyribonucleotide biosynthesis and
nucleotide catabolism
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Ribonucleotide reductase reduces ADP, GDP, CDP
UDP to the deoxyribonucleotides
ribonucleotide reductase
Two cysteines in the enzyme serve as the
reductant.
NADP
NADPH
DNA replication also requires thymine, which is
made from dUDP.
Regeneration of the reduced enzyme requires
several steps
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Each of the active sites in ribonucleotide
reductase has a binuclear Fe center and a stable
tyrosyl radical
tyrosyl radical
This is the E. coli enzyme, in which generating
the Tyr radical requires O2. The enzymes from
some anaerobic bacteria use a B12 derivative or
Fe-S center to generate a radical.
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Ribonucleotide reductase generates free-radical
intermediates
1
NDP
OH-
The tyrosine radical generates another radical
(X?) closer to the substrate.
2
H
3
dNDP
Most electron-transfer reactions of NADH involve
hydride ion transfer.
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Two chains of redox proteins carry electrons from
NADPH to ribonucleotide reductase (RNR)
RNR
glutaredoxin
glutathione
Glutathione (g-Glu-Cys-Gly) is the main redox
buffer in the cytosol of most eukaryotic cells
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Regulation of ribonucleotide reductase at two
allosteric sites balances the rates of formation
of the different deoxyribonucleotides
dTTP inhibits reduction of CDP UDP, and
activates reduction of GDP.
ATP activates reduction of CDP UDP.
allosteric effectors
ATP, dATP, dGTP, dTTP
dATP inhibits and ATP activates the enzyme
with all substrates.
ADP, CDP, UDP, GDP
substrates
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At the activity-regulation site, dATP inhibits
RNR and ATP increases Vmax for the reactions of
all the ribonucleotides
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At the specificity site, ATP, dATP, dTTP and dGTP
tune the relative affinities of RNR for CDP, UDP,
GDP and ADP

ATP
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Thymidylate (dTMP) can be synthesized from either
CDP or UDP
dUMP
N5,N10-methylene-THF
thymidylate synthase
dTMP
Hydrolysis of dUTP looks wasteful. Why dont
cells use dUTP to make dTTP directly?
ATP
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Thymidylate synthase oxidizes N5,N10-methylenetetr
ahydrofolate to dihydrofolate
N5,N10-methylene-tetrahydrofolate
7,8-dihydrofolate
dTMP
dUMP
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Dihydrofolate reductase regenerates
tetrahydrofolate
dihydrofolate reductase
7,8-dihydrofolate
tetrahydrofolate
NADPH H NADP
Serine
serine hydroxymethyl transferase (PLP)
Glycine
N5,N10-methylene-tetrahydrofolate
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Enzymes of nucleotide biosynthesis provide
targets for cancer chemotherapy
methotrexate
Analog of DHF. Inhibits dihydrofolate reductase
(in vivo)
Analog of dUMP. Inhibits thymidylate synthase.
5-fluorouracil
FdUMP
Analog of Gln. Inhibits glutamine
amidotransferases (steps 1 4 of purine
biosynthesis, CTP synthase, carbamoylphosphate
synthetase II).
azaserine (O-diazoacetyl-L-serine)
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Primates oxidize purines to uric acid for
excretion
uric acid
adenosine
guanosine
Birds, reptiles and insects also excrete uric
acid. Fish and most terrestrial mammals oxidize
uric acid further before excreting the product.
uric acid has several tautomeric forms
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Uric acid production from purines
adenosine
inosine
hypoxanthine
adenosine deaminase
O2 H2O
xanthine oxidase
H2O2
xanthine
guanosine
uric acid
O2 H2O
Xanthine oxidase contains FAD, a Mo complex, and
two Fe-S centers. Its substrates are the free
purines, not the nucleosides or nucleotides.
H2O2
xanthine oxidase
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Mutations in adenosine deaminase cause Severe
Combined Immunodeficiency Disease
deoxy-adenosine
uric acid
adenosine deaminase
In the absence of adenosine deaminase, dATP
accumulates to high levels. dATP inhibits
ribonucleotide reductase, which prevents
synthesis of other deoxyribonucleotides by
cutting off DNA synthesis. Lymphocytes are
particularly susceptible to this inhibition.
Untreated SCID is fatal. SCID was the first
human disorder to be addressed by gene therapy.
dATP
-
see Lehninger, Box 9-2 (p. 336)
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Deposition of sodium urate crystals in tissues
causes gout
Impaired excretion of uric acid results in high
levels of uric acid in body fluids. Crystals of
sodium urate form in the toe, kidney and other
tissues, causing painful inflamation.
Gout can be treated with allopurinol, an analog
of hypoxanthine that inhibits xanthine oxidase.
sodium urate
allopurinol
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Salvage pathways regenerate nucleotides from
free purine and pyrimidine bases
adenine phosphoribosyl transferase
adenine
PPi
AMP
Another enzyme (hypoxanthine-guanine
phosphoribosyl transferase) works on hypoxanthine
and guanine. Mutations in this enzyme lead to
Lesch-Nyhan syndrome.
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Lesch-Nyhan syndrome
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Lesch-Nyhan Syndrome can be diagnosed prenatally
Fetal fibroblasts obtained by amniocentesis are
cultured in the presence of 3H-hypoxanthine.
Cells from normal individuals convert
hypoxanthine into IMP, which procedes to AMP and
GMP and is incorporated into DNA.
normal
Lesch-Nyhan
hypoxanthine
IMP
Blocked in Lesch-Nyhan syndrome