3. Know why several valuable anticancer drugs block
the synthesis of thymidylate
• Rapidly dividing cells require an abundant supply of
thymidylate for the synthesis of DNA.
• The vulnerability of these cells to the inhibition of TMP
synthesis has been exploited in cancer chemotherapy.
• Thymidylate synthase and dihydrofolate reductase are
choice targets of chemotherapy.
FATIMA BASHER & MURTAZA
MUHAMMED
3
4. CONT…..
• Fluorouracil, a clinically useful anticancer drug, is
converted in vivo into fluoro-deoxy uridylate.
• This analog of dUMP irreversibly inhibits thymidylate
synthase after acting as a normal substrate through part
of the catalytic cycle.
• Recall that the formation of TMP requires the removal of
a proton (H+) from C-5 of the bound nucleotide.
FATIMA BASHER & MURTAZA
MUHAMMED
4
5. CONT…….
• the enzyme cannot abstract F+ from F-dUMP, and so
catalysis is blocked at the stage of the covalent complex
formed by F-dUMP, methylenetetrahydrofolate, and the
sulfhydryl group of the enzyme.
• The synthesis of TMP can also be blocked by inhibiting
the regeneration of tetrahydrofolate. Analogs of
dihydrofolate,
• such as aminopterin and methotrexate (amethopterin),
are potent competitive inhibitors (K i< 1 nM) of
dihydrofolate reductase.
FATIMA BASHER & MURTAZA
MUHAMMED
5
6. CONT…….
• Methotrexate is a valuable drug in the treatment of many
rapidly growing tumors, such as those in acute leukemia
and choriocarcinoma, cancer derived from placental
cells.
• However, methotrexate kills rapidly replicating cells
whether they are malignant or not.
• Stem cells in bone marrow, epithelial cells of the
intestinal tract, and hair follicles are vulnerable to the
action of this folate antagonist, accounting for its toxic
side effects, which include weakening of the immune
system, nausea, and hair loss.
FATIMA BASHER & MURTAZA
MUHAMMED
6
7. CONT…….
• Folate analogs such as trimethoprim have potent
antibacterial and antiprotozoal activity.
• Trimethoprim binds 105-fold less tightly to mammalian
dihydrofolate reductase than it does to reductases of
susceptible microorganisms.
• Small differences in the active-site clefts of these
enzymes account for their highly selective antimicrobial
action.
• The combination of trimethoprim and sulfamethoxazole
(an inhibitor of folate synthesis) is widely used to treat
infections.
FATIMA BASHER & MURTAZA
MUHAMMED
7
8. Conceive that thymidylate is formed
by the methylation of deoxyuridylate
• Uracil, produced by the pyrimidine synthesis pathway, is
not a component of DNA. Rather, DNA contains thymine,
a
• methylated analog of uracil. Another step is required to
generate thymidylate from uracil. Thymidylate synthase
catalyzes
• this finishing touch: deoxyuridylate (dUMP) is methylated
to thymidylate (TMP)
20XX Yassmin hamada & Mohamed huzayfa 8
9. Cont…
• the methylation of this nucleotide
facilitates the identification of DNA
damage for repair and, hence, helps
preserve the
• integrity of the genetic information
stored in DNA. The methyl donor in
this reaction is N 5,N 10-
• methylenetetrahydrofolate rather
than S-adenosylmethionine.
20XX presentation title 9
10. Cont…
• This methylene group, in turn, is activated by
• distortions imposed by the enzyme that favor opening
the open five-membered ring. The activated UMP's
attack on the
• methylene group forms the new carbon-carbon bond.
The intermediate formed is then converted into product:
a hydride
• ion is transferred from the tetrahydrofolate ring to
transform the methylene group into a methyl group, and
a proton is
• abstracted from the carbon atom bearing the methyl
group to eliminate the cysteine and regenerate the
aromatic ring
20XX presentation title 10
11. Cont...
• Thus, the tetrahydrofolate derivative loses both its
methylene group and a hydride ion and, hence, is
oxidized to
• dihydrofolate. For the synthesis of more thymidylate,
tetrahydrofolate must be regenerated.
20XX Yassmin hamada . Mohamed huzaifa 11
12. Define key steps in nucleotide biosynthesis that are
regulated by feedback inhibition
• Nucleotide biosynthesis is regulated by feedback
inhibition in a manner similar to the regulation of amino
acid biosynthesis.
• Indeed, aspartate transcarbamoylase, is one of the key
enzymes for the regulation of pyrimidine biosynthesis in
bacteria.
• Recall that ATCase is inhibited by CTP, the final product
of pyrimidine biosynthesis, and stimulated by ATP.
• Carbamoyl phosphate synthetase is a site of feedback
inhibition in both prokaryotes and eukaryotes.
18 OCT HANI UMM & REEM MOHAMED 12
13. CONT…..
• The synthesis of purine nucleotides is controlled by
feedback inhibition at several sites
• 1. The committed step in purine nucleotide biosynthesis
is the conversion of PRPP into phosphoribosylamine by
glutamine phosphoribosyl amidotransferase.
• This important enzyme is feedback-inhibited by many
purine ribonucleotides.
• It is noteworthy that AMP and GMP, the final products of
the pathway, are synergistic in inhibiting the
amidotransferase.
HANI UMM & REEM MOHAMED 13
14. CONT…..
• 2. Inosinate is the branch point in the synthesis of AMP
and GMP.
• The reactions leading away from inosinate are sites of
feedback inhibition.
• AMP inhibits the conversion of inosinate into
adenylosuccinate, its immediate precursor.
• Similarly, GMP inhibits the conversion of inosinate into
xanthylate, its immediate precursor.
HANI UMM & REEM MOHAMED 14
15. CONT…….
• 3. As already noted, GTP is a substrate in the synthesis of
AMP, whereas ATP is a substrate in the synthesis of GMP.
• This reciprocal substrate relation tends to balance the
synthesis of adenine and guanine ribonucleotides.
• The reduction of ribonucleotides to deoxyribonucleotides is
precisely controlled by allosteric interactions.
• Each polypeptide of the R1 subunit of the aerobic E. coli
ribonucleotide reductase contains two allosteric sites: one of
them controls the overall activity of the enzyme, whereas the
other regulates substrate specificity.
HANI UMM & REEM MOHAMED 15
16. CONT…….
• The overall catalytic activity of ribonucleotide reductase
is diminished by the binding of dATP, which signals an
abundance of deoxyribonucleotides.
• The binding of ATP reverses this feedback inhibition.
• The binding of dATP or ATP to the substrate-specificity
control sites enhances the reduction of UDP and CDP,
the pyrimidine nucleotides.
HANI UMM & REEM MOHAMED 16
17. CONT……
• The binding of thymidine triphosphate (TTP) promotes
the reduction of GDP and inhibits the further reduction of
pyrimidine ribonucleotides.
• The subsequent increase in the level of dGTP stimulates
the reduction of ATP to dATP.
• This complex pattern of regulation supplies the
appropriate balance of the four deoxyribonucleotides
needed for the synthesis of DNA.
HANI UMM & REEM MOHAMED 17
20. Explain how deoxyribonucleotides synthesized by the
reduction of ribonucleotides through a radical mechanism
• Deoxyribonucleotides are derived from their corresponding
ribonucleotides by direct reduction at the 2’-carbon atof of D-
ribose to form 2’-deoxy derivative of its. The reaction in
which the AD is reduced to 2’-deoxyadenosine diphosphate
occurs in a nonactivated carbon, catalyzed by ribonucleotide
reductase.
• The reduction of the D-ribose portion of a ribonucleoside
diphosphate to 2′-deoxy-D-ribose requires a pair of hydrogen
atoms, which are ultimately donated by NADPH via an
intermediate hydrogen-carrying protein, thioredoxin.
20XX Nisanur Celik 20
21. Continuation
• The ribonucleoside diphosphate is transported by pairs
of —SH groups in thioredoxin from NADPH. Thioredoxin
reductase catalyzes the reduction of its oxidized form by
NADPH, which is subsequently utilized by ribonucleotide
reductase to convert the nucleoside diphosphates into
deoxyribonucleoside diphosphates. Glutathione serves
as a secondary source of reducing equivalents for
ribonucleotide reductase. A protein called glutaredoxin,
which is closely linked to thioredoxin, uses glutathione
as a reductant and then passes the reducing power to
ribonucleotide reductase.
20XX Nisanur Celik 21
22. Continuation
20XX Nisanur celik 22
Glutaredoxin or thioredoxin
transports electrons from
NADPH to the enzyme. Two
molecules of attached
glutathione contribute the
sulfide groups in glutaredoxin
reductase.
The flavoenzyme,
thioredoxin reductase, has
the prosthetic group FAD.
23. Continuation
• The regulation of E. coli ribonucleotide reductase is
remarkable in that binding of effector molecules controls
both the enzyme's activity and substrate specificity. Each
subunit has two different types of regulatory sites. One
type influences overall enzyme activity and binds either
ATP or dATP, activating or inactivating the enzyme. In
reaction to the effector molecule—ATP, dATP, dTTP, or
dGTP—that is bonded there, the second type modifies
substrate specificity. Reduction of UDP and CDP is
induced when ATP or dATP is bound.
20XX Nisanur Celik 23
24. Continuation (last part)
• The plan is made to offer a source of DNA synthesis
precursors balanced. Additionally, ribonucleotide
reduction and biosynthesis are both generally activated
by ATP. The degradation of pyrimidine nucleotides is
accelerated by the tiny levels of dATP present. High
levels of dTTP signify an overstock of pyrimidine dNTPs,
which changes the specificity to favor GDP reduction.
20XX Nisanur Celik 24
