2004 Lec 42-43: Nucleotide Metabolism

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Transcript 2004 Lec 42-43: Nucleotide Metabolism

Biosynthesis and Degradation
of Nucleotides
Lecture 42-43
Baynes & Dominiczak, Chapter 28
Gene C. Lavers, Ph.D.
[email protected]
©Copyright 1999-2004 by Gene C. Lavers
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Purine and Pyrimidine
Parent heterocyclic compounds
Nucleic Acid Metabolism
Fig. 28.1 Structure of purines and pyrimidines.
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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Purine and Pyrimidine Family
Bases, nucleosides, nucleotides
D + O2 
Nucleic Acid Metabolism
 N9-H  N– + H+
basic N
 Purines and pyrimidines are semi-
aromatic ring systems
 p-p bonds similar to benzene
 Bases stack vertically
 Ribose in RNA; deoxyribose in DNA
 (d)Base-sugar = (d)nucleoside
 (d)Base-sugar-PO4= = (d)nucleotide
 Base-pairs
 A = T or T = A
A=U or U=A
 C  G or G  C
 A C G U are in RNA
 dA dC dG dT are in DNA
Fig. 28.2 Names of purine and pyrimidines.
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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Metabolic Roles
Nucleotides
Nucleic Acid Metabolism
Introduction
 DNA and RNA Synthesis
 Protein Synthesis
 Glycogen Synthesis
 Oxidative Phosphorylation
 Signal Transduction
 Muscle Contraction
 Electrolyte Balance
 Coenzymes
 Allosteric regulators
dNTP, NTP
ATP, GTP
UDP-glucose
ADP/ATP
cAMP cGMP
ATP
ATP
NAD FAD CoA
ATP, AMP …
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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Purine synthesis
Ten steps ( 5 + 5)  IMP
Nucleic Acid Metabolism
Cytoplasm
PRPP used in other Rx
1.
1PRaPP + NH2  PRbNH2
2.
Amide with gly; ATP used
3.
1-carbon (C8) N10-THFA
4.
Gln  amidine (N3)
5.
Ring-closure – H2O
6.
CO2 (C6)
7.
Asp (N1) [i.e., urea cycle]
8.
 fumarate
9.
1-carbon (C2) N10-THFA
10.
Ring-closure – H2O
Fig. 28.3 Metabolic pathway for
synthesis of purines
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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IMP  AMP and GMP
Branched pathway
D + O2 
Nucleic Acid Metabolism
Cytoplasm
 IMP common precursor for AMP and GMP
 AMP
– GTP (GDP) cross-substrate for AMP synthesis
– Adenylosuccinate similar to urea cycle compound
–  fumarate + AMP
 GMP
– Oxidation (NAD+) yields xanthine-5’P (XMP)
– ATP (AMP + PP2Pi) amination (gln) amide yields GMP
 Energy cost
– IMP synthesis costs 4 ATP
– AMP synthesis costs GTP (or 5 high energy bonds)
– GMP synthesis costs ATP (or 6 high energy bonds)
 Purine synthesis is proportional: A > G
 Phosphorylations
– AMP  ADP  ATP
– GMP  GDP  GTP
Fig. 28.4 Conversion of IMP to AMP and GMP.
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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Purine synthesis
Control and Salvage of IMP, AMP and GMP
Nucleic Acid Metabolism
Cytoplasm
Control of Purine Synthesis
 Product inhibition by AMP and GMP from IMP
 Cross-nucletide co-substrates required  balanced synthesis
 Allosteric feedback inhibition of PRPP-Gln amidotransferase
Salvage of free bases
 Nucleosides and nucleotides spontaneously hydrolyze N-b-glycosidic bond  free
base released  catabolized to urate
 De novo synthesis of purines minimized by 1-step conversion back to nucleoside-5’P
– Adenine
+ PRPP  AMP
– Hypoxanthine + PRPP  IMP
– Guanine
+ PRPP  GMP
via A-PRTase
via H-PRTase
H-PRTase
Gout and Lesch-Nyhan syndromes
 Salvage enzymes deficient (0.1% in brain gives Lesch-Nyhan psychiatric behavior)
– Up to 5-fold extra de novo synthesis leads to excessive accumulation of urate.
– Urate is sparingly soluble; needle crystals form in joints and kidneys  gouty arthritis and
kidney damage. Untreated L-N children die in teenage years – renal damage failure.
 Treatment: inhibition (X) of XO by allopurinol prevents excessive purine base
catabolism to urate, i.e., ade  hyp —X xan —X urate (Fig 28.5), thereby
limiting excessive purine synthesis that occurs in untreated patients
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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Azaserine
Diazo-containing Antibiotics
Purine Nucleotide Biosynthesis
Clinical
1. Glutamine analogs as affinity labels.
Azaserine and 6-diazo-5-oxo-norleucine
2. Diazo-containing antibiotics bind in active site of amidotransferase.
Diazo group alkylation of nucleophilic cysteine residue in enzyme’s
active site.
3. Expect many enzymes with Gln as substrate would be inactivated by
diazo-containing antibiotics.
O
O
H 2N
NH2
glutamine
O
OH N=N=C
H
O
O
azaserine
NH2
O
OH N=N=C
H
O
OH
NH2
6-diazo-5-oxo-norleucine
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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Catabolism of Purines
Nucleic Acid Metabolism
Allopurinol treatment of gout and Lesch-Nyhan syndromes
Fig. 28.5 Inhibition of xanthine oxidase (XO) by
alloxanthine is the mechanism involved in
allopurinol treatment of gout and LeschNyhan syndromes.
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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Pyrimidine Synthesis
Multifunctional enzymes, CAD and UMP synthase
Nucleic Acid Metabolism
 CP made by carbamoyl phosphate synthetase


different from CPS that makes CP in urea cycle.
In prokaryotes individual enzymes
In eukaryotes 3 + 2 multifunctional enzymes
 Dihydroorotate dehydrogenase linked to ETS via



ubiquinone  2 ATP.
Dihydroorotate oxidized to orotate by
mitochondrial enzyme.
PPRP + orotate  orotate nucleotide  UMP + CO2
UMP  UDP  UTP (gln) CTP
Fig. 28.6 Metabolic pathway for the synthesis of
pyrimidines.
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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Biosynthesis of CTP and TTP
THFA vs. DHFA and chemotherapy
Nucleic Acid Metabolism
Cytosol
Fig. 28.7 Synthesis of
pyrimidine
triphosphates.
Inhibited at the indicated
sites by
• fluorodeoxyuridylate
(FdUMP)
• methotrexate
• aminopterin
• trimethoprim
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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Ribose to Deoxyribose
Nucleic Acid Metabolism
Cytoplasm
Ribonucleotide reductase
H+ + NADPH
Thioredoxin reductase
NADP+
FAD FADH2
Glutathione reductase
Glutathione
Thioredoxin
Glutaredoxin
Ribonucleotide reductase
PPO
Base
O
PPO
2
Base
O
2
OH H
OH OH
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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Uracil to Thymine: 1-carbon fragment on folate
Dihydrofolate: dead-end metabolite
Deoxy Nucleic Acids
Enzymology
Fudr (fluorodeoxyuridate)
cytoplasm
Suicide inhibitor
dUMP
TMP synthase
1
FH2
N5,N10-CH2-FH4
glycine
3. Serine
hydroxymethyl
transferase
serine
dTMP
NADPH + H+
2. Dihydrofolate
reductase
FH4
Rib5P
Pentose
Shunt
NADP+
aminopterin
Glc6P
methotrexate
(amethopterin)
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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DHFA Reductase
Deoxy Nucleic Acids
Enzymology
Chemotherapy
cytoplasm
N
H 2N
N
H
N
5
N
dUMP dTMP
H
Thymidylate synthase
HO H C N10
2
N5N10methyleneTHFA
N
H 2N
H
N
R
HO
Dihydrofolate
DHFA
5
N
N
H
10
(Glu)
O
O
H 2N
N
NH2
N
(Glu)n
5
N
N
H 2N
H
N
N
HN
10
R = H, aminopterin
R = CH3, methotrexate
N
(glu)n
OCH3
OCH3
OCH3
NH2
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
trimethoprim
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Deoxyribose and Thymine
Nucleic Acid Metabolism
Overview
cytoplasm
ribose
deoxyribose
NMP
NDP
Ribonucleoside
bisphosphate
reductase
pp
NTP
dUMP
TMP synthase
DNA
dCTP
DNA
dNTP
dNDP
1
dATP
dGTP
DNA
dUTP
2
dTMP
dTTP
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
DNA
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Synthesis, Catabolism, Salvage
C.P.
Asp
Pyrimidine Metabolism
Overview
3 steps
Orotate
1 enzyme
2 Pi
PRPP
PPi
UMP
“OMP”
CO2
Glycogen
UDP
UTP
dUDP
dUMP
Cyd
dTMP
Urd
dTTP
Thd
RNA
CTP
?
Cyt
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
Ura
b-Ala
Thy
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2-MeBu
Biosynthesis and Degradation
of Nucleotides
END
©Copyright 1999-2004 by Gene
C. Lavers, Ph.D.
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