Nucleotide Synthesis

1. In the synthesis of IMP, why is the second reaction the first committed step? What other
pathways utilize PRPP?
The first step is the synthesis of PRPP. This is not the committed step because PRPP can be
utilized in other pathways, one being the synthesis of histidine along with ATP.
2. How is the purine synthetic pathway controlled?
The committed step is controlled by products AMP and GMP. This step is the amidotransferase
of amine to PRPP.
Inosinate, the branch point of AMP and GMP, can be controlled. AMP inhibits the conversion to
adenylosuccinate, its immediate precursor. GMP inhibits the conversion to xanthylate.
GTP is a substrate in the synthesis of AMP while ATP is a substrate in synthesis of GMP. This
reciprocal substrate relation tends to balance the synthesis of adenine and guanine
ribonucleotides.
3. Where does the NH2 come from to form AMP & GMP from IMP?
4. Describe the different mechanisms of amination of IMP to form GMP or AMP.
In the synthesis of AMP, aspartate is first condensed with inosinate to form adenylosuccinate. It
then breaks off as fumarate leaving behind an amine.
In the synthesis of GMP, XMP, an intermediate, receives an amine from glutamine which turns
glutamine to glutamate.
5. How does UMP synthesis differ in prokaryotes and eukaryotes?
In bacterial cells, aspartate transcarbamoylase is the key enzyme for the regulation of pyrimidine
biosynthesis. This protein is inhibited by CTP. Also the carbamoyl phosphate synthetase is a site
of feedback inhibition. In bacteria, the CPS uses glutamine as a source of amine. The reaction is
then catalyzed by aspartate transcrabamoylase. Removal of water from N-carbamoylaspartate is
catalyzed by dihydroorotase to form dihydroorotate. This is then oxidized to orotate using
NAD+. Orotate is attached to PRPP to yield orotidylate. This is decarboxylated to give uridylate.
In eukaryotes, the first three enzymes in this pathway, carbamoyl phosphate synthetase II,
aspartate transcarbamoylase, and dihydroorotase, are part of a single trifunctional protein known
as CAD.
6. How is prymidine biosynthesis regulated in bacteria and eukaryotic animal cells?
In bacteria, it is largely through ATCase which catalyzes the first reaction in the sequence and
inhibited by CTP, the end product of the sequence. Bacterial ATCase contain six catalytic
subunits and six regulatory subunits. The molecule exists in two conformations, active and
inactive. When CTP is not bound to the allosteric sites, the enzyme is maximally active. ATP
prevents the changes induced by CTP.
UTP can bind to allosteric sites but only inhibits when combined with CTP. It leads to enhanced
affinity for CTP at the high affinity sites.
Reason for this probably to balance the purines and pyrimidines.
Animal ATCase is not regulatory but is controlled by CPS II. CPSII domain is activated by ATP
and inhibited by UDP, UTP, dUTP, and CTP. Role of glycine in ACTase regulation is to act as a
competitive inhibitor of the glutamine binding site.
7. Describe the evolutionary changes that occur to the proteins pyrimidine pathway
structurally from prokaryotes to eukaryotes?
See 5 and 6
8. What is the role of CPSII in UMP synthesis in eukaryotic cells?

See 5 and 6
9. Describe the unusual proton transfer from dihydrooratedh to CoQ that occurs in
eukaryotes.
CAD binds to the mitochondrial outer membrane which allows for formation of dihydroorotate.
It is located in the cytosol. CAD can position itself near the mitochondriaand translocate along
the filmanet to the mitochondria where DHOdhase is located. The physical association of CAD
with the mitochondriais an attractive idea because under physiological conditions, carbamoyl
aspartate is favored over dihydroorotate. Docking allows more efficient capture of
dihydroorotate by DHOdhase and prevent the accumulation of carbamoyl aspartate. The
electrons are transferred to coenzyme q in the mitochondrial membrane via FMN protein
DHOdhase.
10. How does the ribosylation of IMP differ from UMP?
11. Discuss the structure of ribonucleotide reductase. Describe the active site located on the
protein? Describe the mechanism of forming the deoxy-nucleotide.
See 12
12. Describe the interaction between the activity site and the specificity site in ribonucleotide
reductase?
Ribonucleotide reductase consists of two subunits: R1 and R2. Each contain two subunits each.
R1 contains the active site as well as three independent regulatory binding sites that control the
enzyme. R1 catalytic residues tontain several redox active thiol groups.
R2 contains a novel binuclear Fe(III). The Fe complex with tyr 122 forms an unusual tyr-O free
radical.
The activity site binds either ATP or dATP with low affinity where as the specificity site bind
ATP, dATP, dGTP, or dTTP with high affinity. The binding with low affinity ensures that dNTP
synthesis does not stop right away. Binding of ATP at activity site increases enzymatic activity
while dATP decreases it.
If ATP/dATP is bound to specificity site, dUTP and DCTP is favored. Binding of TTP promotes
the reduction of GDP and inhibits further pyrimidine reduction. dGTP stimulate reduction of
ATP.
13. How do the activity site and the specificity site affect the substrate-binding site
ribonucleotide reductase?
See 12
14. What is the role of thioredoxin and thioredoxin reductase in maintaining the activity of
ribonucleotide reductase?
Thioredoxin reduces ribonucleotide reductase so that it can then be used to reduce NDP to dNDP.
Thioredoxin itself is reduced by the action of thioredoxin reductase, a flavoprotein which
receives its electrons from NADPH.

15. Discuss the regeneration of N5N10MTHF from the serine-glycine complex and the
methylation of dUMP to form dTMP.
Serine becomes glycine whiel THF gains a methylene group as well as a hydride ion. THF
therefore becomes oxidized to dihydrofolate. THF is regenerated by dihydrofolate reductase with
the use of NADPH as the reductant. THF is turned to N5N10MTHF.
16. Describe the purine nucleotide cycle and its role in skeletal muscle.
17. Describe the role of HGPRT and APRT in recycling purines.
HGPRT salvages hypoxanthine and PRPP to make IMP and PPi as well as GMP. APRT salvages
adenine and PRPP to make AMP and PPi.
18. How is HGPRT the major deletion mutation that is central to forming a hybridoma that
produces a monoclonal antibody?
Select for HGPRT + monoclonal cell lines. This can be done by using a HAT medium containing
hypoxanthine, aminopterin, and thymidine. Aminopterin inhibits the enzyme dihydrofolate
reductase, necessary to regenerate THF which is later used to methylate dUMP. Without the de
novo synthesis, cells can only rely on salvage pathways to make thymidine. Plasma cells also
eventually die since they are not immortal. Hybrid cells continue to live and divide.