Download LECT 35 Deoxy

5’-AMP
5’-IMP
ADP
ATP
dADP
5’-XMP
dATP
5’-GMP
GDP
dGDP
dGTP
GTP
5’-UMP
UDP
UTP
dUDP
CTP
CDP
dUTP
dCMP
dCDP
dUMP
dCTP
Rapid
dTTP
dTDP
dTMP
Formation of
Deoxyribonucleotides
Two Component System to Reduce Ribose
1. Enzyme: ribonucleotide reductase
2. Electron transport system: thioredoxin or glutaredoxin
Methylation of 5’-dUMP
1. Methylene THF
2. Thymidylate synthase
Ribonucleoside Reductase
Tetramer (4 subunits) 22
The two  subunits are part of protein R1
The two  subunits are part of protein R2
R1 has the catalytic site
3 cysteines (catalytic site)
2 cysteines (redox active site)
R2 has a tyrosine free radical with 2 Fe3+
Redox Site
R1 (2)
-SH HS-
-SH HS-
SH
-SH HS-
SH
-SH HS-
2 allosteric sites for
ATP, dATP, dGTP, dTTP
Catalytic Site
O
O
R2 ( 2)
Fe
Fe
O
Fe
Fe
O
Two identical pairs of subunits
Binuclear Fe(III)
with Tyrosine
Free radical
Formation of Deoxyribonucleotides
Enzyme
E-X
H
H
H
H
H
HO
E-X-H
OH
S-H
E
S-H
E-X +
+OH
HO
H
S
E
SH
H
H
HO
H
Deoxyribose
HO
+
+ H2O
H
S
E
S
E-X-H
H
HO
H
Hydride ion
Regeneration of Sulfhydryls Groups in
Ribonucleotide Reductase
SH
SH
E
S
TR
S
SH
TR
SH
E
SH
R
SH
S
R
S
FAD
FADH2
NADPH
NADP+
S
S
Ribonucleotide
Reductase
Thioredoxin
Thioredoxin
Reductase
Where are the Electrons
NADPH
FAD
TR SH
SH
dNDP
rNDP
reductase
S
NADP
FADH2
S
TR S
S
Thioredoxin
reductase
NADPH
NADP
Glutathione
reductase
2GS-SG
2GSH
SH
GR SH
rNDP
reductase
S
HS
GR S
Glutaredoxin
SH
rNDP
Catalytic Specificity
• One enzyme is used for ADP, GDP, CDP, UDP
(dTMP is formed via dUMP)
• Balance is maintained by end products (dNTP’s)
• Activity sites and Specificity sites
• ATP increases efficiency for ALL substrates
• dATP decreases efficiency of all substrates
• Specificity site maintains balance of the 4 dNTP’s
• dTTP activates GDP but inhibits UDP, CDP
Post Synthesis modifications
5’-UMP
UDP
UTP
Glutamine
O
dUDP Glutamate
HN
O
Deoxy Thymidine
synthesis requires
THF
CTP
N
dUMP
R
5,10-Methylene THF
O
HN
O
CH3
N
R
Dihydrofolate
dTMP
Thymine
dUMP
dTMP
Thymidylate
Synthase
N
H2C
CH2
N
N
CH2
N
H
Glycine
NADPH + H+
H
Serine
N
H
CH2
N
H
TS and DHFR
are targets of anti-cancer drugs
NADP+
Dihydrofolate
reductase
Enzyme commits suicide
by attacking substrate
O
HN
O
F
PRPP
5-fluoro-dUMP
(5-fluorodeoxyuridylate)
N
5-fluorouracil
N
O
HN
Suicide Inhibitor
p713
O
CH2
CH2
NH
F
N
H
S- Enzyme
Enzyme unable to extract F
Nucleoside Analogs are the Basis of Anticancer and
Antiviral Drugs
Why nucleosides?
Because they penetrate membranes more readily
Because they react with internal kinases
Antiviral
HOCH2
HO
araA (arabinosyladenine)
araC (arbinosylcytosine)
R
HO
Arabinose
R=adenine or cytosine
See page 713
Folic Acid
N
H2N
N
H
O
N
N
CH2
OH
NH2
N
H
CH
COO
H
C N C H
CH2
3
CH2
Aminopterin
Anti-Leukemia agents
COO
Methotrexate
Inhibitors of dihydrofolate
reductase
blocks deoxythymidine synthesis
Dideoxynucleosides
R
HOCH2
2’3’-Dideoxycytidine
2,3’-Dideoxyinosine
H
H
H
H
O
CH3
HN
O
HOCH2
- +
N=N=N
N
3’Azido-2’,3’-dideoxythymidine
O
H
AZT
Quiz 3
Take out a Sheet of paper and number its
from 1-7. You will be given 5 minutes to
identify by name the factor involved in the
following metabolic scheme
1
2 NH3
N2
2
-Kg
NADPH
1
NH
PRPP
5
3
NADP+
3
Glutamate
3-phosphohydroxypyruvate
3-phospho7
serine
-Kg
2
Glutamine
4
3
Glutamate
PPi
5-phospho6
ribosylamine