Transcript nucleicacidmetabolism

Nucleic Acid
Metabolism
Andy Howard
Introductory Biochemistry
6 May 2008
Nucleic Acid Metabolism
06 May 2008
What we’ll discuss

Pyrimidine synthesis
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PRPP
Pathway to UMP
Regulation
Pathway to CTP
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Purine synthesis
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IMP
AMP, XMP, GMP
Regulation
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Nucleic Acid Metabolism
Reduction of
riboNucs to
deoxyNucs
dUMP to dTMP
Salvage pathways
Pyrimidine
catabolism
Purine catabolism
p.2 of 56
06 May 2008
Phosphoribosyl
pyrophosphate
PRPP
synthetase
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Activation of ribose-5-P
(see Calvin cycle, etc.)
by ATP:
-ribose-5-P + ATP 
PRPP + AMP
Has roles in other
systems too
Nucleic Acid Metabolism
PRPP synthetase
PDB 2H06
215 kDa hexamer
dimer shown; human
p.3 of 56
06 May 2008
Pyrimidine synthesis:
carbamoyl aspartate
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Uridine is based on orotate,
which is derivated from
carbamoyl aspartate
We’ve already seen the
carbamoyl phosphate synthesis
back in chapter 17 via carbamoyl
phosphate synthetase
Carbamoyl phosphate +
aspartate carbamoyl aspartate
+ Pi
via aspartate transcarbamoylase
Nucleic Acid Metabolism
p.4 of 56
Carbamoyl
phosphate
Carbamoyl
aspartate
06 May 2008
Aspartate
transcarbamoylase
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ATCase is the classic
allosteric enzyme
E.coli version is inhibited
by pyrimidine nucleotides
and activated by ATP
CTP by itself is 50%
inhibitory; CTP+ UTP is
almost totally inhibitory
Nucleic Acid Metabolism
p.5 of 56
ATCase
PDB 1D09
Trimer of
heterotetramers
1 heterotetramer
shown (cf.
fig.18.11)
E.coli
06 May 2008
Carbamoyl aspartate
to dihydroorotate

Dihydroorotate

Carbamoyl
aspartate
dehydrates and
cyclizes to Ldihydroorotate
via
dihydroorotase
TIM barrel protein
Nucleic Acid Metabolism
p.6 of 56
PDB 1XGE
76 kDa dimer
E.coli
06 May 2008
Dihydroorotate to
orotate

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Ubiquinone acts as
oxidizing agent
reducing the 5 & 6
Carbons via
dihydroorotate
dehydrogenase
Some versions
incorporate FMN
Nucleic Acid Metabolism
p.7 of 56
PDB 2E6F
69 kDa dimer
Trypanosoma
cruzi
06 May 2008
Adding
phosphoribose
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Orotidine 5’monophosphate
Orotate + PRPP 
orotidine 5’monophosphate +
PPi
Usual argument re
pyrophosphate
hydrolysis
Enzyme: orotidine
phosphoribosyl
transferase
Nucleic Acid Metabolism
p.8 of 56
PDB 2PS1
50 kDa dimer
Yeast
06 May 2008
Decarboxylation
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OMP
decarboxylated to
form UMP via OMP
decarboxylase
Bacterial forms are
TIM barrel proteins
Acceleration is
1017-fold relative to
uncatalyzed rate
Nucleic Acid Metabolism
p.9 of 56
PDB 1KLY
54 kDa dimer
Methanobacterium
thermoautotrophicum
06 May 2008
Eukaryotic variation
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Orotate produced in the
mitochondrion moves to the
cytosol
UMP synthase combines the
last two reactions—orotidine
to OMP to UMP
Nucleic Acid Metabolism
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OMP
decarboxylase
domain
of UMP synthase
PDB 2P1F
64 kDa dimer
human
06 May 2008
UMP to UTP
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Uridylate kinase converts
UMP to UDP:
UMP + ATP  UDP + ADP
enzyme is related to
several amino acid kinases
Nucleoside diphosphate
kinase exchanges di for tri:
UDP + ATP  UTP +ADP
(non-specific enzyme)
Nucleic Acid Metabolism
p.11 of 56
Uridylate kinase
PDB 2A1F
163 kDa hexamer
Haemophilus
influenzae
06 May 2008
CTP synthetase
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UTP + gln + ATP 
CTP + glu + ADP + Pi
Glutamine side-chain is
amine donor
ATP provides energy
 sandwich (Rossmann)
Enzyme is inhibited by CTP
In E.coli, it’s activated by GTP
(makes sense!)
Nucleic Acid Metabolism
p.12 of 56
PDB 1S1M
240 kDa tetramer
dimer shown
E.coli
06 May 2008
Purine synthesis
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Considerably more complex than
pyrimidine synthesis
More atoms to condense and two rings to
make
More ATP to sacrifice during synthesis
Several synthetase (ligase) reactions
require ATP
Based on PRPP, gln, 10-formyl THF, asp
Nucleic Acid Metabolism
p.13 of 56
06 May 2008
PRPP + gln to
phosphoribosylamine
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1
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PRPP aminated:
PRPP + gln  glu + PPi +
5-phospho--Dribosylamine
via glutamine-PRPP
amidotransferase
transferase structure
Product is unstable
(lasts seconds!)
Nucleic Acid Metabolism
p.14 of 56
PDB 1ECF
120 kDa tetramer
dimer shown
E.coli
06 May 2008
Phosphoribosylamine
to GAR
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2
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Amine condenses with
glycine to form
glycinamide
ribonucleotide (GAR)
ATP hydrolysis drives
GAR synthetase
reaction to the right PDB 2YRX
50 kDa monomer
Geobacillus kaustophilus
Nucleic Acid Metabolism
p.15 of 56
06 May 2008
FGAR
Formylation
of GAR
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3
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10-formyl THF donates
a formyl (-CH=O) group
to end nitrogen with the
help of GAR
transformylase to form
formylglycinamide
ribonucleotide (FGAR)
Rossmann 
Nucleic Acid Metabolism
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PDB 1MEO
47 kDa dimer
human
06 May 2008
FGAR to
FGAM
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4
Glutamine sidechain
is source of N for
C=O exchanging to
C=NH via FGAM
synthetase to form
formylglycinamidine
ribonucleotide
(FGAM):
FGAR + gln + ATP +
H2O  FGAM + glu
+ ADP + Pi
PurS component of
FGAM synthetase
PDB 1GTD
37.4 kDa tetramer
dimer shown
Methanobacterium
Nucleic Acid Metabolism
p.17 of 56
FGAM
06 May 2008
Aminoimidazole
ribonucleotide
FGAM to AIR
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5
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Cyclize FGAM to
aminoimidazole
ribonucleotide
ATP drives the AIR
synthetase reaction:
FGAM + ATP 
AIR + H2O + ADP + Pi
E.C. in Wikipedia is
wrong:
it should be 6.3.3.1
Nucleic Acid Metabolism
PDB 2V9Y
147 kDa tetramer
dimer shown
human
p.18 of 56
06 May 2008
CAIR
AIR to CAIR
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6
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AIR is carboxylated;
expenditure of an ATP:
AIR + HCO3- + ATP 
carboxyaminoimidazole
ribonucleotide + ADP +
Pi + 2H+
AIR carboxylase
E.coli version is two
enzymes; eukaryotes
have a single enzyme
No cofactors!
Nucleic Acid Metabolism
p.19 of 56
PDB 2NSH
149 kDa octamer
monomer shown
E.coli
06 May 2008
CAIR+asp to
SAICAR
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7
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CAIR + asp + ATP 
aminoimidazole
succinylocarboxamide
ribonucleotide + ADP + Pi
Enzyme is SAICAR
synthetase
Domain 1: homolog of
phosphorylase
kinase
Domain 2: ATP-binding
Nucleic Acid Metabolism
p.20 of 56
PDB 2CNQ
34 kDa
monomer
yeast
06 May 2008
SAICAR to AICAR
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8
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SAICAR  aminoimidazole
carboxamide ribonucleotide
+ fumarate
Enzyme is adenylosuccinate
lyase
Net result of two reactions is
just replacing acid with
amide;
That’s like first 2 reactions in
urea cycle, except ADP, not
AMP, is the product
Nucleic Acid Metabolism
p.21 of 56
PDB 2PTR
203 kDa tetramer
dimer shown; E.coli
06 May 2008
AICAR to FAICAR
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9
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10-formylTHF donates HC=O:
AICAR + 10-formylTHF 
formamidoimidazole
carboxamide ribonucleotide +
THF
Enzyme: AICAR transformylase
Like step 3
Generally a bifunctional enzyme
combined with next step
This part is like cytidine
deaminase (see below)
Nucleic Acid Metabolism
p.22 of 56
PDB 1THZ
130 kDa dimer
chicken
06 May 2008
FAICAR to IMP
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10
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We made it:
FAICAR  inosine 5’monosphosphate + H2O
Bifunctional enzyme;
this part is called IMP
cyclohydrolase or
inosicase
Hydrolase part is like
methylglyoxal synthase
Nucleic Acid Metabolism
PDB 1PL0
260 kDa tetramer
dimer shown; human
p.23 of 56
06 May 2008
So now we have a
purine. What next?
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Enzymatic conversions to AMP or GMP;
Details on next few slides
AMP and GMP can be further
phosphorylated to make ADP, GDP with
specific kinases (adenylate kinase and
guanylate kinase)
GTP made with broad-spectrum
nucleoside diphosphate kinase
Nucleic Acid Metabolism
p.24 of 56
06 May 2008
IMP to
adenylosuccinate
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IMP + aspartate + GTP 
adenylosuccinate + GDP + Pi
Enzyme is adenylosuccinate
synthase
Similar to step 7 in IMP
synthesis
PDB 2V40
101 kDa dimer
monomer shown
human
Nucleic Acid Metabolism
p.25 of 56
06 May 2008
Adenylosuccinate
to AMP

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Adenylosuccinate 
AMP + fumarate
Like reaction 8 in the
IMP pathway; in fact, it
uses the same enzyme,
adenylosuccinate lyase
PDB 2PTR
203 kDa tetramer
dimer shown; E.coli
Nucleic Acid Metabolism
p.26 of 56
06 May 2008
IMP to XMP
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IMP + H2O + NAD+ 
Xanthosine
monophosphate +
NADH + H+
Enzyme: IMP
dehydrogenase
TIM-barrel, aldolase- PDB 1ME8
like protein
221 kDa tetramer;
monomer shown
Tritrichomonas foetus
Nucleic Acid Metabolism
p.27 of 56
06 May 2008
XMP to GMP
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XMP + gln + H2O +
ATP  GMP + glu +
AMP + PPi
Enzyme: GMP
synthetase
Typical 3-layer
sandwich
Nucleic Acid Metabolism
PDB 2DPL
68 kDa dimer
Pyrococcus horikoshii
p.28 of 56
06 May 2008
Adenylate
kinase
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Reminder:
ATP + AMP  2 ADP
Metal ions play a role in
enzyme structure
Enzymes like this need
to shield their active sites
from water to avoid
pointless hydrolysis of
ATP
Nucleic Acid Metabolism
PDB 1ZIN
24 kDa monomer
Bacillus
stearothermophilus
p.29 of 56
06 May 2008
Guanylate kinase
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GMP + ATP  GDP + ADP
“P-loop”-containing ATPbinding proteins
Rossmann fold
PDB 2QOR
22 kDa monomer
Plasmodium vivax
Nucleic Acid Metabolism
p.30 of 56
06 May 2008
Purine control I: IMP level
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Note that GTP is a cosubstrate in making
AMP from IMP
ATP is a cosubstrate in making GMP
from IMP
So this helps balance the 2 products
Nucleic Acid Metabolism
p.31 of 56
06 May 2008
Purine control II:
feedback inhibition
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PRPP synthetase inhibited by purines, but
only at unrealistic concentrations of [Pur]
Step 1 (gln-PRPP amidotransferase) is
allosterically inhibited by IMP, AMP, GMP
Adenylosuccinate synthetase is inhibited
by AMP
XMP and GMP inhibit IMP dehydrogenase
Nucleic Acid Metabolism
p.32 of 56
06 May 2008
Making
deoxyribonucleotides
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Conversions of nucleotides to
deoxynucleotides occurs at the
diphosphate level
Reichard showed that most organisms
have a single ribonucleotide reductase
that converts ADP, GDP, CDP, UDP to
dADP, dGDP, dCDP, and dUDP
NADPH is the reducing agent
Nucleic Acid Metabolism
p.33 of 56
06 May 2008
RNR1
PDB 1R1R
258 kDa
dimer
E.coli
Ribonucleotide
reductase
heterotetramer
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2 RNR1 subunits; each has
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a helical 220-aa domain
10-strand 480-aa structure
(thiols here)
5-strand 70-aa structure
RNR2
PDB 1PJ0
82 kDa
dimer
E.coli
2 RNR2 subunits; each has
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A diferric ion center
A stable tyrosyl free radical
Nucleic Acid Metabolism
p.34 of 56
06 May 2008
Mechanism of RNR
(box 18.3)
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Y122 in RNR2 is converted to stable free
radical
Radical transmitted to RNR1 cys439
Cys439 reacts with substrate 3’-OH to form
free radical at C3’
Substrate dehydrates to carbonyl at C3’ and
free radical at C2’; S- formed at Cys462
Disulfide formed between Cys462,Cys225;
radical regenerated at Cys439
Nucleic Acid Metabolism
p.35 of 56
06 May 2008
Ribonucleotide
reductase: control

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ATP, dATP, dTTP, and dGTP act as allosteric
modulators by binding to two regulatory sites
on the enzyme
Activity site (A) regulates activity of catalytic
site
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When ATP binds at A, activity goes up
When dATP binds at A, activity inhibited overall
Specificity site (S) controls which substrates
can be turned over
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ATP at A + ATP or dATP at S : pyrimidines only
dTTP at S : activates reduction of GDP
dGTP at S : activates reduction of ADP
Nucleic Acid Metabolism
p.36 of 56
06 May 2008
dUDP to dUMP
(for making dTMP)

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dTMP formed at monophosphate
level
(from dUMP)
dUMP derived three ways:

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
dUDP + ADP  dUMP + ATP
dUDP + ATP  dUTP + ADP
dUTP + H2O  dUMP + PPi
dCMP + H2O  dUMP + NH4+
Nucleic Acid Metabolism
p.37 of 56
06 May 2008
Thymidylate synthase
reaction (fig.18.15)
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dUMP + 5,10-methyleneTHF 
dTMP + 7,8-dihydrofolate
dihydrofolate
Unusual THF reaction in that cofactor gets
oxidized as well as giving up a carbon
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5,10-methylene
THF
CH2 from 5,10-methylene group
extra H from C6
So DHF must be reduced back to THF via
DHFR and get its methylene back from SHMT
Nucleic Acid Metabolism
p.38 of 56
06 May 2008
Thymidylate
synthase
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Generally the controlling step in
DNA synthesis because [dTTP] <
other [deoxynucleoside
triphosphates]
Therefore a target for cancer
chemotherapy and other therapies
that target rapidly-dividing cells
Enzyme is a 2-layer sandwich
Nucleic Acid Metabolism
p.39 of 56
PDB 2G8O
58 kDa dimer
E.coli
(with dUMP
and cofactor
analog)
06 May 2008
Thymidylate synthase
and drug design

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Both folate analogs and dUMP
analogs can interfere with
(DHFR  SHMT  dTMP
synthase  … ) cycle
5-fluorouracil is specific to
thymidylate synthase
Nucleic Acid Metabolism
p.40 of 56
06 May 2008
DHFR
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Converts DHF to THF:
DHF + NADPH + H+ <->
THF + NADP+
SHMT then converts THF to
5,10-methyleneTHF
3-layer  sandwich
Often the target for drug design
as well
Eukaryotic DHFR also
catalyzes folate  DHF
Prokaryotic DHFR doesn’t;
DHF derived by another
mechanism in bacteria
Nucleic Acid Metabolism
p.41 of 56
PDB 1KMV
20 kDa monomer
human
folate
06 May 2008
Special case:
protozoan
TSynth/DHFR

Bifunctional enzyme:
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Thymidylate synthase
Dihydrofolate reductase
Presumably some entropic
advantage
Maybe electrostatics too,
allowing the negative charges
on DHF to tunnel through;
but cf. Atreya et al (2003)
J.Biol.Chem. 278:28901.
Nucleic Acid Metabolism
p.42 of 56
DHFR-TS
PDB 1J3K
104 kDa
dimer
Plasmodium
falciparum
06 May 2008
Recovery
pathway to
dTMP

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Deoxythymidine can be
phosphorylated by thymidine
kinase:
deoxythymidine + ATP 
dTMP + ADP
Labeled thymidine is
convenient for monitoring
intracellular synthesis of DNA
because thymidine enters cells
easily
Nucleic Acid Metabolism
p.43 of 56
PDB 1E2K
73 kDa
monomer
Herpes
simplex virus
06 May 2008
Fates of
polynucleotides

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Nucleic acids hydrolyzed to
mononucleotides via nucleases
Mononucleotides are dephosphorylated via
nucleotidases and phosphatases
Resulting nucleosides are deglycosylated
via nucleosideases or nucleoside
phosphorylases
Resulting bases are sent either into salvage
pathways or get degraded and excreted
Nucleic Acid Metabolism
p.44 of 56
06 May 2008
Salvage pathways

We can describe them, and we will: but
why do they matter so much?

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They provide energy savings relative to de novo
synthesis (think of all the ATP we used in
making IMP!)
Considerable medical significance to
interference with these pathways
Intracellular nucleic acid bases are usually
recycled; dietary bases are usually broken
down and excess nitrogen excreted
Nucleic Acid Metabolism
p.45 of 56
06 May 2008
Orotate
phosphoribosyl
transferase

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
Principal salvage enzyme for
pyrimidines
Orotate + PRPP -> OMP + PPi
OMP can then reenter UMP
synthetic pathway
(decarboxylation to UMP, then
form UDP and CDP)
Same enzyme can aact on other
pyrimidines to make nucleotides:
Pyr + PRPP -> PyrMP + PPi
Nucleic Acid Metabolism
p.46 of 56
PDB 2 PS1
50 kDa dimer
Yeast
06 May 2008
Pyrimidine interconversions
(fig. 18.19)

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All phosphorylations & dephosphorylations can
and do happen
UTP can be aminated to CTP
CDP and UDP can be reduced to dCDP and
dUDP
dCMP can deaminate to dUMP
Cytidine can be converted to uridine
dUMP can be methylated to dTMP
Nucleic Acid Metabolism
p.47 of 56
06 May 2008
Purine
nucleotide
salvage



Two phosphoribosyl
transferases convert adenine,
guanine, and hypoxanthine to
AMP, GMP, and IMP
Adenine phosphoribosyl
transferase is specific
HGPRT accepts both
hypoxanthine and guanine
Nucleic Acid Metabolism
p.48 of 56
Hypoxanthineguanine
phosphoribosyl
transferase
PDB 1FSG
102 kDa tetramer
dimer shown
Toxoplasma gondii
06 May 2008
Purine Interconnections
(fig. 18.18)

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All phosphorylations and
dephosphorylations can and do occur
ADP and GDP can be reduced to dADP
and dGDP
AMP can deaminated to IMP (new)
IMP can be aminated to AMP
IMP can oxidized to XMP
XMP can be aminated to GMP
Guanine, adenine can be
phosphoribosylated to GMP and AMP
Nucleic Acid Metabolism
p.49 of 56
06 May 2008
Fates of CMP
and cytidine


CMP’s phosphate can be
hydrolyzed off
That’s followed by
deamination of cytidine to
make uridine


Catalyzed by cytidine
deaminase
Another sandwich
protein
Nucleic Acid Metabolism
p.50 of 56
Cytidine deaminase
PDB 2FR5
64 kDa tetramer
Mouse
06 May 2008
Hydrolysis of
U, dU and dT

Glycosidic bond in uridine or
thymidine is hydrolyzed by
phosphate:




Uridine + Pi -> -D-ribose-1-P +
uracil
Enyzme is uridine
phosphorylase
Similar enzyme handles
deoxyuridine
Similar reaction using
thymidine phosphorylase
yields thymine + -Ddeoxyribose-1-P
Nucleic Acid Metabolism
p.51 of 56
Uridine
phosphorylase
PDB 1RXY
167 kDa
hexamer
Dimer shown
E.coli
06 May 2008
Uracil to acetyl CoA;
thymine to succinyl CoA




Reduced to dihydrouracil and
dihydrothymine
Hydrated and ring-opened to
ureidopropionate or
ureidoisobutyrate
Eliminate bicarbonate and
ammonium to yield -alanine or aminoisobutyrate
Several reactions from there to
acetyl CoA and succinyl CoA
Nucleic Acid Metabolism
p.52 of 56
Dihydropyrimidinase
PDB 1GKP
302 kDa
hexamer
Thermus
06 May 2008
Uric acid
Purine catabolism


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
Nucleoside or deoxynucleoside +
phosphate  base + (D)-ribose 1-P
Hypoxanthine and guanine both lead to
uric acid as a product
Uric acid is the final excreted
nitrogenous compound in primates and
birds and some reptiles
Other organisms catabolize it further
Nucleic Acid Metabolism
p.53 of 56
06 May 2008
Uric acid
Uric acid to
allantoin



Urate oxidase:
urate + 2H2O + O2 
allantoin + H2O2 + CO2
That’s the final product in a
lot of mammals, turtles,
some insects, gastropods
Other organisms catabolize
allantoin further; we’ll talk
about that on Thursday
Nucleic Acid Metabolism
p.54 of 56
Allantoin
Urate oxidase
134 kDa tetramer
monomer shown
Aspergillus flavus
06 May 2008
Lesch-Nyhan
syndrome




Michael
Lesch
William
Nyhan
Complete lack of hypoxanthine-guanine
phosphoribosyl transferase
So hypoxanthine and guanine are degraded
to uric acid rather than being built back up into
IMP and GMP
Leads to dangerous buildup of uric acid in
nervous tissue
Neurological effects are severe and poorly
understood
Nucleic Acid Metabolism
p.55 of 56
06 May 2008
Sodium
urate
Gout


Sodium urate
crystals
accumulating
Accumulation of sodium urate and
uric acid, both of which are only
moderately soluble
Arises from inadequate (~10%)
functionality of HGPRT, so that
urate accumulates in peripheral
tissues, particularly the feet
Nucleic Acid Metabolism
p.56 of 56
Benjamin
Franklin
(celebrated
gout sufferer)
06 May 2008