Download - 12.12.08: Nucleotide Metabolism
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M1 Renal: Nucleotide Metabolism
Fall 2008
Dr. Robert Lyons Assistant Professor, Biological Chemistry
Director, DNA Sequencing Core
Web: http://seqcore.brcf.med.umich.edu/mcb500
Glu, Gln,Asp, NH3
Amino Acid metabolism Folate metabolism
Nucleic Acid metabolism
MethyleneTHF
MetCycle
Pu rin e s Py rim id in e s
Uric Acid
Urea
Amino acids
DNARNA
(energy)
TCA Cyclefumarate
oxaloacetate
R. Lyons
O MPIM P
U ric A cid
NH4( ene rg y )
CarbamoylPhosphate
PRPP
Nucleic Acid metabolismam ino a c id s ,
fo la te
P u r in e M P P y r im id in e M P
Ribonucleotidereductase
dNTP
Ribonucleotidereductase
dNTPDNANTPRNANTP
PurineBiosynthesis
PyrimidineBiosynthesis
PurineDegradation
PyrimidineDegradation
PurineSalvage
PyrimidineSalvage
Click on any blue rectangle to see details.
R. Lyons
O
OH OHOH
O
ribose - 5 - phosphateATP
O
OH OHO
phosphoribose - 1pyrophosphate
OP
OP P
AMP
P
Formation of PRPP: Phosphoribose pyrophosphate
PRPP Use in Purine Biosynthesis:
O
OH OHO
phosphoribose - 1pyrophosphate
OP
OP Pglutamine
H OO
OH OH
OP NH2
glutamate
2
R. Lyons
R. Lyons
The First Purine: Inosine Monophosphate
N
NN
NH
O
ribose-5-P
GTP GDP + Pi
aspartate fumarate
N
NN
NH
ribose-5-P
N
Inosine monophosphate Adenosine monophosphate
2
Conversion to Adenosine:
Conversion to Guanosine:
N
NN
NH
O
ribose-5-P
NADH + H+
N
NN
ribose-5-P
N
Inosine monophosphate Xanthosine monophosphate
NAD+ O
OH
AMP + PPATP i
gln glu
H O2
N
N
ribose-5-P
N
O
NH
Guanosine monophosphate
N
H
2
O
OH OH
OP
N
NN
NH
O
(folates are involved in this synthesis)
R. Lyons
R. Lyons
R. Lyons
AMP + ATP <--> 2ADP (adenylate kinase) GMP + ATP <---> GDP + ADP (guanylate kinase) • similar enzymes specific for each nucleotide • no specificity for ribonucleotide vs. deoxyribonucleotide
Nucleoside Monophosphate Kinases
Ribonucleotide Reductase
Hydroxyurea inhibits this enzyme: chemotherapeutic use
C O
NH HONH 2
O
OH OH
OP
X
OP
O
OH H
OP
X
OP
NADH
NAD+
Enzyme•
Enzyme
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Regulation of Ribonucleotide Reductase
CDP dCDP dCTP
UDP dUDP dTTP
GDP dGDP dGTP
ADP dADP dATP
+
+
ATP
ATP+
+
(-)
(-)
(-)
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N1DP + N2TP <--> N1TP + N2DP
dN1DP + N2TP <--> dN1TP + N2DP
Nucleoside Diphosphate Kinase
• No specificity for base • No specificity for ribo vs deoxy
Feed-forward regulation by PRPP
PRPP
IMP
GMP AMP
GTP ATP
ATP GTP
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Feed-forward regulation by PRPP
PRPP
IMP
GMP AMP
GTP ATP
ATP GTP
+
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Feed-forward regulation by PRPP
PRPP
IMP
GMP AMP
GTP ATP
ATP GTP+ +
R. Lyons
Feed-forward regulation by PRPP
PRPP
IMP
GMP AMP
GTP ATP
ATP GTP
+- -
--
R. Lyons
N
NN
N
O
H
HNH2
Guanine
H O2NH4
+
ribose
N
NN
NH
N
Adenosine
2
Adenosinedeaminase
(ADA)
H O2 NH4+
N
NN
ribose
N
Inosine
O
H
purine nucleosidephosphorylase
Pi ribose - 1 - PN
NN
N
Hypoxanthine
O
H
H
xanthineoxidase
N
NN
N
O
H
H
Xanthine
HO
xanthineoxidase
N
NN
N
O
H
H
Uric acid
HO
H
O
N
NN
N
O
H
NH2
Guanosineribose
purine nucleosidephosphorylase
Pi ribose - 1 - P
Guaninedeaminase
Degradation of the Purine Nucleosides:
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“Salvage” Pathways for Purine Nucleotides
N
NN
O
HN
HypoxanthineH
O
OH OHO
OP
OP P
PRPP
+ O
OH OH
OP
Inosine monophosphate
N
NN+ PPi
Hypoxanthine-guanine
phosphoribosyltransferase
O
HN
APRT - Adenine phosphoribosyl transferase - performs a similar function with adenine.
R. Lyons
Adenosine Deaminase Deficiency:
Guanine
N
NN
NH
N
Deoxyadenosine
2
Adenosinedeaminase
(ADA)
N
NN
2-deoxyribose
N
Deoxyinosine
O
H N
NN
N
Hypoxanthine
O
H
H
Xanthine
Uric acid
O
OH H
dAMP
HO
dADP
dATP
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N
NN
N
Hypoxanthine
O
H
H
xanthineoxidase
N
NN
N
O
H
H
Xanthine
HO
xanthineoxidase
N
NN
N
O
H
H
Uric acidH
O
H
O
Hyperuricemia can be caused by:
Accelerated degradation of purines:
•Accelerated synthesis of purines •Increased dietary intake of purines
Impaired renal clearance of uric acid
Allopurinol inhibits xanthine oxidase and reduces blood uric acid levels:
N
N
N
OH
Allopurinol
H
N
Gout: deposition of urate crystals in joints, “tophi” in cooler periphery
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An 80-year-old man with a 30-year history of gout, this patient had been treated intermittently to reduce his serum urate levels.
The New England Journal of Medicine
Lesch-Nyhan Syndrome: Defective HGPRT • hyperuricemia • spasticity • mental retardation • self-mutilation behavior
N
NN
O
HN
HypoxanthineH
O
OH OHO
OP
OP P
PRPP
+ O
OH OH
OP
Inosine monophosphate
N
NN+ PPi
Hypoxanthine-guanine
phosphoribosyltransferase
O
HN
A defect in APRT does NOT have similar consequences R. Lyons
Myoadenylate Deaminase ‘Fills’ the TCA Cycle in Muscle
N
NN
NH
N
Adenosine monophosphate
2
N
NN
N
Inosine monophosphate
H
myoadenylatedeaminase
H O2 NH3
Asp, GTP
GDP + Pi
Fumarate
ToTCACycle
O
ribose-5-P ribose-5-P
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2ATP + HCO +glutamine + H O2
3-
C
O
NH O P2
2-+ glutamate + 2ADP + Pi
carbamoylphosphate
Carbamoyl phosphate synthetase II - a cytoplasmic enzyme…
…used for pyrimidine synthesis
N
NH
O
carbamoylphosphate
+
-
aspartate
CO
NH2C
O
OCH2
CHCO2
-NH3+
P2-C
O
NH
O
2
-
C
OO
CH2
CHCO2
-NH
O CO2H
orotate
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O
OH OHO
phosphoribose - 1pyrophosphate
OP
OP P
N
NH
O
O CO2Horotate
+ O
OH OH
OP
N
NH
O
O CO2
orotidinemonophosphate
CO2
O
OH OH
OP
N
NH
O
O
uridinemonophosphate
Orotate is linked to PRPP to form Uridine monophosphate:
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O
OH OH
OP
N
NH
O
O
uridinetriphosphate
gln glu
++H O2 Pi
O
OH OH
OP
N
N
O
cytidinetriphosphate
H2
N
OPOP OPOP
ATP ADP
UTP can be converted to CTP by CTP Synthetase:
Newly-synthesized uridine monophosphate will be phosphorylated to UDP and UTP, as described for the purine nucleotides.
R. Lyons
ATP ADP
dUDP dUTP
PiH O2
dUMPUDPribo-
nucleotidereductase
The Thymidylate Synthase Reaction:
N , Nmethylene
tetrahydrofolate
5 10
dihydrofolate
THFmethylenedonor
NADH
NAD+
DHFR****
O
OH H
OP
N
NH
O
O
deoxyuridinemonophosphate
O
OH H
OP
N
NH
O
O
deoxythymidinemonophosphate
CH3
thymidylate synthase
Some UDP is converted to dUDP via ribonucleotide reductase.
R. Lyons
R. Lyons
Methotrexate Inhibits Dihydrofolate Reductase:
Dihydrofolate builds up, levels of THF become limiting, thymidylate synthase is unable to proceed. Follow it with a dose of Leucovorin, a.k.a. formyl-THF.
N , Nmethylene
tetrahydrofolate
5 10
dihydrofolate
THFmethylenedonor
NADH
NAD+
DHFR****
O
OH H
OP
N
NH
O
O
deoxyuridinemonophosphate
O
OH H
OP
N
NH
O
O
deoxythymidinemonophosphate
CH3
thymidylate synthase
X Methotrexate
R. Lyons
FdUMP Inhibits The Thymidylate Synthase Reaction:
N , Nmethylene
tetrahydrofolate
5 10
dihydrofolate
THFmethylenedonor
NADH
NAD+
DHFR****
O
OH H
OP
N
NH
O
O
5-fluorodeoxyuridinemonophosphate
(F-dUMP)
O
OH H
OP
N
NH
O
O
deoxythymidinemonophosphate
CH3
thymidylate synthase
F
X
R. Lyons
de-novo synthesis OMP
UMP
UDP
UTP
dUDP
dUMP
dUTP
dTMP
dTDP
dTTPCTP
CDPdCDP
dCTP
***
******
***
Complicated Pathways for Pyrimidine Production:
This figure is primarily a study aid; you do not need to memorize it or reproduce it. The information here merely summarizes material from previous sections.
R. Lyons
Pathologies of pyrimidine nucleotide biosynthesis:
Orotic acidurea due to OTC deficiency - please review your Urea Cycle notes.
Hereditary orotic acidurea - deficiency of the
enzyme that convert orotate to OMP to UMP. Not common.
Pyrimidine degradation:
Cytidine deaminase converts cytidine to uridine A phosphorylase removes the sugar Degradation of the base proceeds (products are
unimportant here)
O
OH OH
Cytidine
NO
N
HO
NH2
cytidinedeaminase O
OH OH
Uridine
NO
HN
O
HO
O
OH H
HO
deoxyribose-1-phosphate
+Pi
NH
O
HN
thyminepyrimidine
phosphorylase
O
O P
CH3
+O
OH H
(deoxy)thymidine
NO
HN
O
HO
CH3
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R. Lyons
Pyrimidines can be salvaged as well:
Thymine + deoxyribose-1-phosphate --> thymidine (NOT thymidine monophosphate!)
Thymidine + ATP --> thymidine monophosphate
Enzyme: Pyrimidine nucleoside phosphorylases
Enzyme: Thymidine kinase - adds the monophosphate back
Herpes Simplex Virus carries its own tk gene
O
OH H
HO
deoxyribose-1-phosphate
+Pi
NH
O
HN
thyminepyrimidine
phosphorylase
O
O P
CH3
+O
OH H
(deoxy)thymidine
NO
HN
O
HO
CH3
R. Lyons
Certain drugs act via the pyrimidine salvage pathway:
O
OH H
HO
deoxyribose-1-phosphate
+
NH
O
HN
5-fluorouracil
O
O P
F
O
OH H
fluorodeoxyuridine
NO
HN
O
HO
F
pyrimidinephosphorylase
O
OH H
fluorodeoxyuridine monophosphate(FdUMP)
NO
HN
O
O
F
P
uridinekinase
GO
H
HO
H
Acyclovir
HSVthymidine
kinase
GO
H
P O
H
AcycloGMP
R. Lyons
R. Lyons
DPD inhibitors can potentiate 5FU activity
O
OH H
HO
deoxyribose-1-phosphate
+
NH
O
HN
5-fluorouracil
O
O P
F
O
OH H
fluorodeoxyuridine
NO
HN
O
HO
F
pyrimidinephosphorylase
O
OH H
fluorodeoxyuridine monophosphate(FdUMP)
NO
HN
O
O
F
P
uridinekinase
5-FU --> --> FdUMP + methylene-THF + Thymidylate Synthase
--> inactivation of TS
Degradation (via dihydropyrimidine dehydrogenase, DPD
5-FU efficacy depends on rate of degradation vs activation
R. Lyons
Capecitabine mode of action:
NH
O
HN
fluorouracil
O
FdealkylationO
OH OH
capecitabine
HO
NO
N
HN
OCH3
O
F
cytidinedeaminase
pyrimidinephoshorylase
O
OH
OH
Cytosine arabinoside (araC)
NO
N
HO
NH2
cytidinedeaminase
cytidinekinase O
OH
OH
NO
N
O
NH2
PO
OH
OH
Uridine arabinoside(INACTIVE)
NO
HN
O
HO
Cytosine arabinoside monophosphate(ACTIVE)
Cytosine arabinoside (araC) activation and inactivation:
R. Lyons
R. Lyons
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