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TUTORIAL SLIDES
Pyrimidine synthesis de novo pathway
___ is the sugar substrate for de novo synthesis of pyrimidine nucleotides
(like purine nucleotide synthesis)
Unlike de novo purine nucleotide synthesis, the pyrimidine ring is synthesized ___ and is then attached to PRPP
__ ___ ___ __ catalyzes the formation of carbamoyl phosphate from HC03-, ATP, and glutamine
Carbamoyl phosphate and aspartate are
covalently attached cyclized and modified
to generate ____
Orotate is covalently linked to PRPP to make ___, which is modified to make ___ & ___
Synthesis of deoxyribonucleotides
Nucleotide ___, e.g. GDP, are the substrates to generate deoxynucleotide
diphosphates, e.g. dGDP
___ ___ catalyzes the reduction of NDPs to generate dNDPs. The oxygen on the 2' carbon of the ribose is removed to convert it into ___
This results in production of DP, dGDP, dCDP and dUDP. dADP, dGDP and dCDP
are phosphorylated to become dATP, dGTP and dCTP. dUDP is dephosphorylated to
dUMP. What about dTTP?
The dUMP ring is methylated (methyl comes from __) to generate dTMP, which is phosphorylated twice to form dTTP
Pyrimidine vs purine nucleotide synthesis
• Purines and pyrimidines can be made via de novo and salvage pathways
___ is used as a precursor in both de novo pathways
In purine synthesis, the purine nitrogenous BASE is built from scratch WHILE attached to Cl of the sugar, i.e. PRPP is used in the __ step
In pyrimidine synthesis, the pyrimidine nitrogenous base is built from scratch and THEN attached to Cl of the sugar, i.e. PRPP is used in the ___ step
Pyrimidine nitrogenous bases
The pyrimidine nitrogenous base that is built from scratch is called ___
Orotate reacts with PRPP to generate orotidine 5'- monophosphate (__)
OMP is modified to produce uridine 5 '-monophosphate (___), which in turn is modified to produce the ___ pyrimidine nucleotides
De novo synthesis of pyrimidines
Pyrimidines have 2 nitrogens:
I comes from the side chain of ___ and 1 comes from ____
Pyrimidines have 4 carbons:
I comes from __ and the other 3 come from the ___
De novo synthesis of pyrimidines
The first step is the synthesis of ___ ___
• ___ ____ ____ ___ catalyzes the transfer of the side-chain nitrogen of glutamine to bicarbonate in a reaction requiring 2 ATPs
This reaction occurs in the ____
Carbamoyl phosphate will be linked to ___
• Contrast with the synthesis of carbamoyl phosphate in the UREA cycle
Carbamoyl phosphate synthetase __ catalyzes the reaction of ammonia
with bicarbonate in a reaction requiring 2 ATPs
Remember this reaction occurs in the ___
Carbamoyl phosphate is linked to ___ in the urea cycle
Synthesis of OMP
Carbamoyl phosphate reacts with aspartate to form ___ ___
Reaction is catalyzed by ___ ____ (ATCase).
This enzyme is very important as a key point of regulation of the pathway
Carbamoyl aspartate (a linear molecule) is cyclized by ____ and then oxidized to produce ____
Orotate reacts with PRPP to produce orotidine 5 '-monophosphate
(____)
Synthesis of OMP
In eukaryotes, steps l, 2 and 3 are
catalyzed by one enzyme that has ___ catalytic sites — increase the efficiency of synthesis
The ___ ___ (__) activity is a key site
of regulation
OMP is decarboxylated to produce Uridine 5'-monophosphate (___)
OMP is converted to UMP by
____
UMP is ____ to form UTP
Synthesis of CTP
CTP is generated by transfer of amide nitrogen from glutamine to ___
Catalyzed by CTP synthetase (___ ___ reaction)
*important point of regulation
Allosteric regulation
___ ___ (__) and ___ ___ are allosterically regulated
CTP ____ the activity of both enzymes
If level of UTP is also high, ___ is further inhibited
___ is an allosteric activator of ATCase (if levels are high want to increase pyrimidines)
___ is an allosteric activator of CTP synthetase (if GTP is high want to increase CTP)
Allosteric regulation of aspartate
transcarbamoylase (ATC)
CTP regulates activity (INHIBITS) by binding
to an ___ site FAR from the
active site of the enzyme
Deoxyribonucleotide synthesis
De novo pathways and most salvage pathways generate ribonucleotides (exception is salvage pathway for ___)
Ribonucleotides are reduced to deoxyribonucleotides by ___ ____
Substrates are ___ (e.g. CDP) and products are ___ (e.g. dCDP)
Electrons to reduce NDPs originate in ___
Regulation is complex
Ribonucleotide reductase
Reduction of nucleotide results in ___ of ribonucleotide reductase.
Ribonucleotide reductase needs to be ___ in order to catalyze another reaction.
Regulation of ribonucleotide reductase
Catalytic site ___ NDPs
Activity site binds ___ or
___ — turns enzyme on
or off
Specificity site binds ATP/dATP or dTTP or dGTP - alters substrate ___ to make different dNDPs
Regulation of ribonucleotide reductase
If levels of dNTPs are high, don't need to make more. __ binds to shut OFF enzyme.
If levels of dNTPs are low, need to make more. dATP is low, so ___ binds to turn enzyme ON.
If levels of purines are high, need to make more pyrimidines. ___/__ binds to alter
substrate specificity to make more __ and ____
If levels of pyrimidines are high, need to make more purines. ___ binds to alter substrate specificity to make more ___.
If levels of dGDP is high, need to make more dADP. ___ binds to alter substrate
specificity to make more dADP.
Production of dNTPs
dADP, dGDP and dCDP are phosphorylated to produce dATP, dGTP and dCTP for DNA synthesis
It is very important the _ is NOT incorporated into DNA
dUDP is dephosphorylated to form dUMP (if dUTP is accidently produced it is
rapidly degraded to ___)
• dUMP is methylated to form dTMP, which is phosphorylated to form ___ and ___ for DNA synthesis
Generation of dTTP from dUMP
___ ___ catalyzes the
transfer of the carbon from 5, 10-methylene THF to dUMP to produce dTMP
When methyl group is transferred
THF is oxidized to dihydrofolate
and must be reduced back to THF
by ___ to be used again (recycle THF)
5 -Fluorouracil
Structure is similar to uracil
Can be converted into a nucleotide
Inhibits ___ ____
This is an anti-cancer drug
Why?
Methotrexate:
This is an inhibitor of ___. Therefore reduces level of THF. This is an anti-cancer Why?
Gemcitabine:
This is a pyrimidine ring analogue — can be converted to nucleotide diphosphate mimic — suicide inhibitor of
___ ___ — anti-cancer drug — why?
Recap I
Carbamoyl phosphate synthetase Il produces carbamoyl phosphate
from ___, ___, & ___
Carbamoyl phosphate reacts with aspartate to produce a product that is
cyclized and oxidized to produce ___
Oratate reacts with PRPP to produce ___, which is decarboxylated to
generate ___
Amide group from glutamine is transferred to UTP to produce ___
CTP, UTP, ATP and GTP ____ regulate enzymes in the de novo pyrimidine synthesis pathway
Recap II
NDPs are reduced by ___ ___ to produce dNDPs
Ribonucleotide reductase binds different nucleotides to regulate activity and substrate specificity
dUMP is methylated using 5,10 methylene THF as a carbon donor to
produce ___
5-fluorouracil, methotrexate, and gemcitabine are anti-cancer drugs
that target ____ production
