deoxyribonucleotidos sintesis y degradacion
TRANSCRIPT
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12. Deoxyribonucleotide biosynthesis andnucleotide catabolism
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dTTP
ADP
GDPCDP
UDP
H2O
NADP+ NADPH
ribonucleotidereductase
X
SH
SH
X
S
S
Ribonucleotide reductase reduces ADP, GDP,CDP & UDP to the deoxyribonucleotides
DNA synthesis also
requires thymine, which
is made from dUDP.
Regeneration of the reduced
enzyme requires several steps
Two cysteines in
the enzyme serveas the reductant.
OHOH
OP -O- P -O-CH2
N
OH
P -O- P -O-CH2O
N
dADP
dGDP
dCDP
dUDP
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QuickTime an d a
TIFF (Uncompressed) decompressorare needed to see this p icture.
1av8.pdb
R2dimer
regulatorysites
R2 R2
O O
R1 R1
The enzyme has two types
of subunits, R1 and R2.
Each of the catalytic sites in ribonucleotide reductasehas a binuclear Fe center and a stable tyrosyl radical
O CHCH2
C=O
NH
O OC
Fe Fe
C
O O CO O
NN
NN
CO O
tyrosylradical
two Fe
atoms with His, Glu & Asp ligands
The catalyticsites are atthe R1-R2interfaces
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Ribonucleotide reductase generates free-radical intermediates
NDP
P -O- P -O-CH2 N
OHOH
O
HOHOH
OP -O- P -O-CH2 N
H H
XH
SH
SH
OH
P -O- P -O-CH2 N
OH
+
XH
S
S
HOH
O
P -O- P -O-CH2 N
H H
X
SH
SH
X
S
S
dNDP
1
2
3
The tyrosine radical
generates another radical(X ) close to the substrate.
Most electron-transfer reactions
of NADH involve hydride ion
transfer.
P -O- P -O-CH2 N
HOH
O
H
Steps 1 & 3: H atom
(H ) transfer
Step 2: hydride ion (H )
transfer or two 1-ereactions
OH-
H+
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Two sets of redox proteins carry electrons fromNADPH to ribonucleotide reductase (RNR)
glutathione
glutathionereductase
glutaredoxinreductase
X
SH
SH
X
S
S
NDP dNDP
S
S
SH
SH
GSSG2 GSH
NADPHNADP
+
glutaredoxin
CO2
H3N-CH-CH2CH2-CO-NH-CH-CO-NH-CH2-CO2
CH2SH+
-
-
Glutathione (g-Glu-Cys-Gly) is
the main redox buffer in the
cytosol of most eukaryotic cells
NADP+NADPH
thioredoxinreductase
S
S
SH
SHthioredoxin
X
SH
SH
X
S
S
RNR
RNR
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Regulation of ribonucleotide reductase at two allosteric sitesbalances 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.
dATP inhibits and
ATP activates the
enzyme with all
substrates.
allostericeffectors
substrates
R2 R2
R1 R1
activityregulation
site
substrate-specificitysite
catalyticsite
ATP, dATP,dGTP, dTTP
ADP, CDP,UDP, GDP
SHSH
HSHS
ATP, dATP
-+
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At the activity-regulation site, dATP inhibits RNR and ATPincreases Vmaxfor the reactions of all the ribonucleotides
R1
substrate-specificitysite
R2
catalyticsite
SHSH
activityregulation
site
ADP ATP
dADP dATP
-
+
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At the specificity site, ATP, dATP, dTTP and dGTP tunethe relative affinities of RNR for CDP, UDP, GDP and ADP
+
-
-
-
-
ATP ADP dADP dATP
GDP dGDP dGTP
UDP dUDP dTTP
CDP dCDP dCTP
+
+
+
+
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H2O
PPi
dUMP
dTMP
thymidylatesynthase
N
CHC
O
HN
CHCO
O
OHOH
-O-P-O-CH2
O-
O
N5,N10-methylene-THF
N
CC
O
HN
CHCO
CH3
dTTPATP
dCTPdeaminase
H2O
NH3
ribonucleotidereductase
nucleosidediphosphate
kinase
C
NH2
N
NCHC
O
CH
CDP dCDP dCTP
ATP ADP
UDP dUDP dUTP
ATP ADP
Thymidylate (dTMP) can be synthesized from either CDP or UDP
Hydrolysis of dUTP looks
wasteful. Why dont cells use
dUTP to make dTTP directly?
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In the process of methylating dUMP, thymidylate synthaseoxidizes N5,N10-methylenetetrahydrofolate to dihydrofolate
dTMP
7,8-dihydrofolateN5,N10-methylene-tetrahydrofolate
dUMP
N
CHC
O
HN
CHCO
OH
-O-P-O-CH2
O-
OO
OH
-O-P-O-CH2
O-
OO
CH3
N
CC
O
HN
CHCO
H
N
H
CH
N
HN
OH
H2N N
N
O
C-NH-(Glu)n
H
H
O
C-NH-(Glu)nN
N
HN
OH
H2N N
N
H
H
H
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Dihydrofolate reductase regenerates tetrahydrofolate,using NADPH as the reductant
N5,N10-methylene-tetrahydrofolate
Serine
Glycine
7,8-dihydrofolate tetrahydrofolateNADPH + H+ NADP+
dihydrofolatereductase
serine hydroxymethyltransferase (PLP)
N
HNN
N
OH
H2N
HN R
H
H
OH
H2N N
N
H
H
NH
N
HN
C
H
H
H
HNHN
HNH2N
OH
N
N
H
H
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Enzymes of nucleotidebiosynthesis provide targets
for cancer chemotherapy
methotrexate
Analog of DHF. Inhibits
dihydrofolate reductase
CO2-
+H3N C H
CH2
O
N=N=CH-C-O-C=O
O
- +
azaserine(O-diazoacetyl-L-serine)
Analog of Gln. Inhibits glutamine
amidotransferases (steps 1 & 4 of
purine biosynthesis, CTP synthase, &
carbamoylphosphate synthetase II).
N
C-FC
O
HN
CHC
O
OH
-O-P-O-CH2O
-
OON
C-FC
O
HNCHC
OH
5-fluorouracil FdUMP
Analog of dUMP. Inhibits
thymidylate synthase.
(in vivo)
H2N-C=O
CO2-
+H3N C H
CH2
CH2
Gln
NCH3
N
HN
NH2
H2N N
NO
C-NH-Glu
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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 excretion.
NH2
C
CN
N
N
CH
C
NHC
ribose
C
CHN
N
N
CH
C
O
NH2N-C
ribose
O
C
CHN
N
C OH
C
NC
HO NH
CCOH
C
NN
C OH
NC
HO NH
uric acid has several
tautomeric forms
C
CHN
C O
CO
NCO N
H
NH
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Uric acid production from purines
C
CHN
N
N
CH
CO
NH2N-C
ribose
C
CHN
N
C OH
C
O
NC
HO NH
uric acidguanosine
adenosine inosine hypoxanthine
NH2
C
CN
N
N
CH
C
NHC
ribose ribose
C
CHN
N
N
CH
C
O
NHCadenosine
deaminase
C
CHN
N
N
CH
C
O
NHC
H
H2O NH4+
Pi ribose-1-P
C
CHN
N
N
CH
CO
NC
HHO
xanthine
O2 + H2O
O2 + H2O
H2O2
H2O2
xanthineoxidase
xanthineoxidase
Xanthine oxidase contains FAD, a
Mo complex, and two Fe-S centers.
Its substrates are the free purines,
not the nucleosides or nucleotides.
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In the absence of adenosine deaminase, dATP accumulates to high levels.
dATP inhibits ribonucleotide reductase, which prevents synthesis of other
deoxyribonucleotides and cuts 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.*
Mutations in adenosine deaminase causeSevere Combined Immunodeficiency Disease
deoxy-
adenosine
NH2
C
CN
N
N
CH
C
NHC
deoxyribose deoxyribose
C
CHN
N
N
CH
C
O
NHCadenosine
deaminase
H2O NH4+
uricacid
dATP
ribonucleotides deoxyribonucleotides
ribonucleotidereductase
DNA
- *see Lehninger, Box 9-2 (p. 336)
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Deposition of sodium uratecrystals in tissues causes gout
allopurinol
C
CHN
N
C
N
C
O
NHC
H
H
Impaired excretion of uric acid results in high levels of uric acid in body fluids. Crystals
of sodium urate form in the toes, kidney and other tissues, causing painful inflamation.
uric acidhypoxanthine xanthine
C
CHN N
C OH
C
O
NC
HO NH
C
CHN
N
N
CH
C
O
NHC
H
C
CHN
N
N
CH
C
O
NC
HHO
O2 + H2O H2O2
xanthine
oxidase
xanthine
oxidase
O2 + H2O H2O2
sodium urate
C
CHN
N
C O-
CO
NC
HO NH
Na+Gout can be treated with
allopurinol, an analog of
hypoxanthine that inhibits
xanthine oxidase.
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Salvage pathways regenerate nucleotides
from free purine and pyrimidine bases
A similar enzyme (hypoxanthine-guanine
phosphoribosyl transferase) works on
hypoxanthine and guanine. Mutations in this
enzyme lead to Lesch-Nyhan syndrome.
adenineNH2
C
CN
N
N
CH
C
NHC
H
adeninephosphoribosyltransferase
PPi
O
O- O-
O
O P O P O-
OHOH
OP O-CH2
5-phosphoribosyl-1-pyrophosphate (PRPP)
NH2
C
CN
N
N
CH
C
NHC
OHOH
OP O-CH2
AMP
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Lesch-Nyhan syndrome
Lesch-Nhyan syndrome, which results from a mutation in the gene forhypoxanthine-guanine ribosylphosphotransferase, is characterized bysevere neurological defects.
mental retardation
self-mutilation
cerebral palsy
elevated uric acid (gout)
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Lesch-Nyhan Syndrome can be diagnosed prenatally
Fetal fibroblasts obtained by amniocentesis are
cultured in the presence of3H-hypoxanthine.
Cells from normal individuals converthypoxanthine into IMP, which procedes to AMP
and GMP and is incorporated into DNA.
Blocked inLesch-Nyhan
syndrome
normal
Lesch-Nyhanhypoxanthine
C
CHN
N
N
CH
C
O
NHC
HIMP
OHOH
OP O-CH2
C
CHN
N
N
CH
C
O
NHC
PRPP PPi
AMP
GMP
DNA