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Z Score,T Score, Percential Rank and Box Plot Graph
Nucleotide Metabolism Pathways
1. Author(s): Dr. Robert Lyons, 2009
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3. M1 Renal:
Nucleotide Metabolism
Dr. Robert Lyons
Assistant Professor, Biological Chemistry
Director, DNA Sequencing Core
Web: http://seqcore.brcf.med.umich.edu/mcb500
Fall 2008
4. Amino Acid metabolism
Folate metabolism
Amino acids
Met
Methylene Cycle
Glu, Gln, THF
Asp, NH 3
Urea
oxaloacetate
DNA
P u rin e s P y rim id in e s
RNA
Uric Acid (energy)
fumarate
TCA Cycle Nucleic Acid metabolism
R. Lyons
5. Nucleic Acid metabolism
Click on any blue rectangle to see details.
am i n o a c id s ,
Carbamoyl
fo la t e
Phosphate
PRPP
Purine
Salvage Purine Pyrimidine OMP
IM P Biosynthesis Biosynthesis
Pyrimidine
Salvage
P u r in e M P P y r im id in e M P
Ribonucleotide Ribonucleotide
reductase reductase
dNTP DNA dNTP
NTP RNA NTP
Purine Pyrimidine
Degradation Degradation
NH4
U ric A cid ( e n e rg y )
R. Lyons
6. Formation of PRPP: Phosphoribose pyrophosphate
O O
PO P O
OH O P O P
OH OH OH OH
ATP AMP
ribose - 5 - phosphate phosphoribose - 1
R. Lyons
pyrophosphate
PRPP Use in Purine Biosynthesis:
H2
O
O O NH2
PO P O
O PO P
OH OH glutamine glutamate OH OH
phosphoribose - 1
pyrophosphate
R. Lyons
7. O
N
HN
The First Purine: Inosine Monophosphate
N N
(folates are involved in this synthesis)
O
P O
OH OH
R. Lyons
Conversion to Adenosine:
O NH 2
GTP GDP + Pi
N N
HN N
N N N N
aspartate fumarate ribose-5-P
ribose-5-P
Inosine monophosphate Adenosine monophosphate
R. Lyons
Conversion to Guanosine:
H2O
+ + O O
O NAD NADH + H ATP AMP + PPi
N N N
HN N HN
N N O N N NH N N
H 2
gln glu
ribose-5-P ribose-5-P ribose-5-P
Inosine monophosphate Xanthosine monophosphate Guanosine monophosphate
R. Lyons
8. Nucleoside Monophosphate Kinases
AMP + ATP <--> 2ADP
(adenylate kinase)
GMP + ATP <---> GDP + ADP
(guanylate kinase)
• similar enzymes specific for each nucleotide
• no specificity for ribonucleotide vs. deoxyribonucleotide
9. Ribonucleotide Reductase
X
O
P OP O
Enzyme• NADH
OH OH
X
O
P OP O
Enzyme NAD+
OH H
R. Lyons
Hydroxyurea inhibits this enzyme: chemotherapeutic use
O
HONH
C
NH
2
10. Regulation of Ribonucleotide Reductase
ATP
CDP + dCDP dCTP
(-)
ATP
UDP + dUDP dTTP
(-)
GDP + dGDP dGTP
ADP + dADP dATP
(-)
R. Lyons
11. Nucleoside Diphosphate Kinase
N1DP + N2TP <--> N1TP + N2DP
dN1DP + N2TP <--> dN1TP + N2DP
• No specificity for base
• No specificity for ribo vs deoxy
16. Degradation of the Purine Nucleosides:
+
NH 2 HO NH O Pi ribose - 1 - P O
2 4
N N N
N HN HN
Adenosine purine nucleoside
N N N N N N
deaminase phosphorylase H
ribose (ADA) ribose
Adenosine Inosine Hypoxanthine
xanthine
oxidase
Pi ribose - 1 - P
O + O
O H 2O NH 4
N N
HN N HN
HN
N purine nucleoside NH Guanine O N N
NH 2 N N N H H
phosphorylase 2
H deaminase
ribose
Xanthine
Guanosine Guanine xanthine
oxidase
O
H
N
HN
Uric acid O
O N N
H H
R. Lyons
17. Salvage Pathways for Purine Nucleotides
O
N O
HN
Hypoxanthine- N
N N guanine HN
H
Hypoxanthine phosphoribosyl
N N
transferase
+ + PPi
O
P O
O
P O
O P O P OH OH
OH OH Inosine monophosphate
PRPP
R. Lyons
APRT - Adenine phosphoribosyl transferase -
performs a similar function with adenine.
18. Adenosine Deaminase Deficiency:
NH 2
N O O
N
N N
HN HN
N N
N N N N
HO O Adenosine H
deaminase 2-deoxyribose
(ADA) Hypoxanthine
Deoxyinosine
OH H
Deoxyadenosine
Guanine Xanthine
dAMP
dADP
Uric acid
dATP
R. Lyons
19. O
HN
N Gout: deposition of urate crystals in joints,
N N tophi in cooler periphery
H
Hypoxanthine
xanthine Hyperuricemia can be caused by:
oxidase
O Accelerated degradation of purines:
HN
N
O N
H
N
H
•Accelerated synthesis of purines
Xanthine
•Increased dietary intake of purines
xanthine
oxidase
Impaired renal clearance of uric acid
O
H
N OH
HN H
O N
N
O N
H
N
H
Allopurinol inhibits xanthine oxidase
N
N
Uric acid and reduces blood uric acid levels:
Allopurinol
R. Lyons
R. Lyons
20. 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
21. Lesch-Nyhan Syndrome: Defective HGPRT
• hyperuricemia
• spasticity
• mental retardation
• self-mutilation behavior
O
N O
HN
Hypoxanthine- N
N N guanine HN
H
Hypoxanthine phosphoribosyl
N N
transferase
+ + PPi
O
P O
O
P O
O P O P OH OH
OH OH Inosine monophosphate
PRPP
R. Lyons
A defect in APRT does NOT have similar consequences
22. Myoadenylate Deaminase Fills the TCA Cycle in Muscle
H 2O NH 3
myoadenylate
deaminase
NH 2
O
N
N N
HN
N N
N N
ribose-5-P ribose-5-P
Adenosine monophosphate Inosine monophosphate
Asp, GTP
Fumarate
GDP + Pi
To
TCA
Cycle
R. Lyons
23.
24. Carbamoyl phosphate synthetase II - a cytoplasmic enzyme…
O
- 2-
2ATP + HCO3 + + glutamate + 2ADP + Pi
NH2 C OP
glutamine + H 2O
carbamoyl
phosphate
R. Lyons
…used for pyrimidine synthesis
O O
- O
O
NH2 -
O C C
CH2 NH2 CH2 HN
C 2-
+
O OP CH C CH O
- - N CO2
NH3 CO2 O N CO2 H
+
carbamoyl H
phosphate aspartate orotate
R. Lyons
25. Orotate is linked to PRPP to form Uridine monophosphate:
O
HN
O O
O N CO2
H HN HN
orotate
O N CO2 O N
+ O
P O O
P O
O
P O CO2
O P OP OH OH OH OH
OH OH orotidine uridine
phosphoribose - 1 monophosphate monophosphate
pyrophosphate
R. Lyons
26. Newly-synthesized uridine monophosphate will be phosphorylated to
UDP and UTP, as described for the purine nucleotides.
UTP can be converted to CTP by CTP Synthetase:
O NH 2
HN N
O N gln glu O N
O O
P OP O P O P OP O P O
OH OH ATP ADP OH OH
+ +
uridine H 2O Pi cytidine
triphosphate triphosphate
R. Lyons
27. Some UDP is converted to dUDP via ribonucleotide reductase.
UDP dUDP dUTP dUMP
ribo-
nucleotide
reductase ATP ADP H 2O Pi
R. Lyons
The Thymidylate Synthase Reaction:
O O
HN HN CH3
O N O N
O O
P O P O
thymidylate synthase
OH H OH H
deoxyuridine 5 10 deoxythymidine
monophosphate N ,N
methylene monophosphate
dihydrofolate
tetrahydrofolate
DHFR NADH
****
methylene THF
NAD+
donor
R. Lyons
28. Methotrexate Inhibits Dihydrofolate Reductase:
O O
HN HN CH3
O N O N
O O
P O P O
thymidylate synthase
OH H OH H
deoxyuridine deoxythymidine
monophosphate N 5, N10
methylene monophosphate
dihydrofolate
tetrahydrofolate
DHFR NADH
**** X Methotrexate
methylene THF
donor NAD+
R. Lyons
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.
29. FdUMP Inhibits The Thymidylate Synthase Reaction:
O O
HN F HN CH3
O N O N
O O
P O P O
thymidylate synthase
OH H
X
OH H
5-fluorodeoxyuridine deoxythymidine
monophosphate N 5, N10
monophosphate
(F-dUMP) methylene dihydrofolate
tetrahydrofolate
DHFR NADH
****
methylene THF
NAD+
donor
R. Lyons
30. Complicated Pathways for Pyrimidine Production:
dCTP CTP UTP dUTP dTTP
***
dCDP CDP UDP dUDP dTDP
*** ***
UMP dUMP dTMP
***
de-novo synthesis OMP
R. Lyons
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.
31. 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.
32. Pyrimidine degradation:
Cytidine deaminase converts cytidine to uridine
NH2 O
HN
O
N
N O N
cytidine
HO
O deaminase HO
O
OH OH OH OH
Cytidine Uridine
R. Lyons
A phosphorylase removes the sugar
O
O HN CH3
O
HN
N
CH3
Pi
pyrimidine
phosphorylase
O N
H
thymine
+
HO
O
HO
+
O OP
OH H
(deoxy)thymidine OH H
deoxyribose-1-phosphate
R. Lyons
Degradation of the base proceeds (products are
unimportant here)
33. Pyrimidines can be salvaged as well:
Enzyme: Pyrimidine nucleoside phosphorylases
Thymine + deoxyribose-1-phosphate --> thymidine
(NOT thymidine monophosphate!)
O
O HN CH3
HN CH3 O N
pyrimidine H
O N Pi phosphorylase thymine
+
O +
HO
O OP
HO
OH H
(deoxy)thymidine OH H
deoxyribose-1-phosphate
R. Lyons
Enzyme: Thymidine kinase - adds the monophosphate back
Thymidine + ATP --> thymidine monophosphate
Herpes Simplex Virus carries its own tk gene
34. Certain drugs act via the pyrimidine salvage pathway:
G HSV G
HO O thymidine P O O
kinase
H H H H
Acyclovir AcycloGMP
R. Lyons
O
HN F O
O
HN F
O N HN F
H pyrimidine O N uridine
5-fluorouracil phosphorylase kinase O N
O
+ HO O
O P O
HO OP
OH H
OH H
OH H
deoxyribose-1-phosphate fluorodeoxyuridine fluorodeoxyuridine monophosphate
(FdUMP)
R. Lyons
35. 5-FU efficacy depends on rate of degradation vs activation
O
HN F O
O
HN F
O N HN F
H pyrimidine O N uridine
5-fluorouracil phosphorylase kinase O N
O
+ HO O
O P O
HO OP
OH H
OH H
OH H
deoxyribose-1-phosphate fluorodeoxyuridine fluorodeoxyuridine monophosphate
(FdUMP)
R. Lyons
5-FU --> --> FdUMP
+ methylene-THF + Thymidylate Synthase
--> inactivation of TS
Degradation
(via dihydropyrimidine dehydrogenase, DPD
DPD inhibitors can potentiate 5FU activity
36. Capecitabine mode of action:
O
CH3
O
HN
N F
O
O N cytidine pyrimidine
dealkylation deaminase phoshorylase
HN F
O
HO O N
H
OH OH fluorouracil
capecitabine
R. Lyons
Cytosine arabinoside (araC) activation and inactivation:
O NH2 NH2
HN
N N
O N
cytidine O N cytidine O N
O deaminase O kinase
HO O
OH HO P O
OH OH
OH OH OH
Uridine arabinoside Cytosine arabinoside (araC) Cytosine arabinoside monophosphate
(INACTIVE) (ACTIVE)
R. Lyons
37. Additional Source Information
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