 Pyrimidine synthesis

 Pyrimidine catabolism
➢ Pyrimidine ring
synthesized before
being attached to ribose
5-phosphate donated by
PRPP
Sources of atoms in
pyrimidine ring
 glutamine
 CO2
 aspartate
Regulated step in
mammalian or eukaryotic
cells :
 Glutamine and CO2
combine to form carbamoyl
phosphate in the presence of
ATP catalyzed by cytosolic
carbamoyl phosphate
synthetase (CPS- II)
Regulation of CPS II :
 Inhibitor: uridine
triphosphate UTP, end
product
 Activator : PRPP
• Cytosolic carbamoyl
phosphate synthetase II
a different enzyme from
the mitochondrial
carbamoyl phosphate
synthetase I of urea
synthesis
• Compartmentation
provides an independent
pool of carbamoyl
phosphate for each
process
 Increased excretion of
orotic acid, uracil and
uridine occurs in a
deficiency in liver
mitochondrial ornithine
transcarbamoylase.
 Excess carbamoyl
phosphate exits to the
cytosol where it stimulates
pyrimidine nucleotide
biosynthesis .
 The resulting mild orotic
aciduria is increased by high
nitrogen foods.
 Five of the first six enzyme activities of pyrimidine
biosynthesis reside on multifunctional polypeptide
 CAD, a single polypeptide named for the first letters of its
enzyme activities, catalyzes the first three reactions.
 A second bifunctional enzyme ,UMP synthase ,catalyzes
further reactions.
 The close proximity of multiple active sites on a
multifunctional polypeptide facilitates efficient channeling of
the intermediates of pyrimidine biosynthesis.
 Carbamoyl Phosphate
Synthetase II

Aspartate
transcarbamoylase

Dihydroorotase

First three enzymic activities in pyrimidine pathway:

three different catalytic domains of a single
polypeptide
Activator : PRPP
Inhibitor : UTP
Second step in pyrimidine
synthesis :
 Aspartate added to
carbamoyl phosphate with
formation of carbamoyl
aspartate, catalyzed by
aspartate
transcarbamoylase
 Regulated step in
prokaryotic cells
 Inhibitor of aspartate
transcarbamoylase in
prokaryotic cells :CTP
 Pyrimidine ring closed
by dihydroorotase
forming
dihydroorotate
 Dihydroorotate oxidized
to produce orotic acid
or orotate by
dihydroorotate
dehydrogenase
 FMN reduced in this
reaction. Pyrimidine
ring formed.
 Dihydroorotate
dehydrogenase, a
flavoprotein associated
with the inner
mitochondrial
membrane.
 All other enzymes in
pyrimidine biosynthesis
are cytosolic
 Completed pyrimidine ring
converted to the nucleotide
orotidine monophosphate
(OMP) by reacting with
PRPP
 Donor of ribose 5-
phosphate: PRPP
 Enzyme :Orotate
phosphoribosyl-
transferase produces OMP
, releases pyrophosphate in
irreversible reaction
 parent pyrimidine
mononucleotide
 OMP decarboxylated to uridine monophosphate
UMP by orotidylate decarboxylase.
 Orotate phosphoribosyltransferase and orotidylate
decarboxylase catalytic domains of a single
polypeptide chain -UMP synthase.
 UMP is sequentially
phosphorylated to UDP
and UTP
 Cytidine triphosphate
(CTP) produced by
amination of UTP by
CTP synthetase with
glutamine providing
the nitrogen
 UDP generates dUDP by
ribonucleotide reductase
 dUDP phosphorylated to
dUTP, which is rapidly
hydrolyzed to dUMP by
UTP diphosphatase or
dUTPase.
 dUTPase plays an important
role in reducing availability
of dUTP as a substrate for
DNA synthesis, preventing
erroneous incorporation of
uracil into DNA.
 Cytidine triphosphate
(CTP) produced by
amination of UTP by
CTP synthetase with
glutamine providing
the nitrogen
➢ CTP is dephosphorylated
to cytidine diphosphate
(CDP), substrate for
ribonucleotide reductase.
 dCDP can be
phosphorylated to dCTP
for DNA synthesis or
dephosphoprylated to
dCMP that is deaminated
to dUMP.
 dUMP converted to
deoxythymidine
monophosphate (dTMP) by
thymidylate synthase
 Source of methyl group:
N5,N10-methylene
tetrahydrofolate
 an unusual reaction in that
tetrahydrofolate (THF)
contributes not only a one-
carbon unit but also two
hydrogen atoms from the
pteridine ring, resulting in the
oxidation of THF to
dihydrofolate DHF
 Thymine analogs :5-
fluorouracil, antitumor
agent
 5-Fluorouracil is
converted to 5-
fluorodeoxyuridine
monophosphate (5-
FdUMP), which becomes
permanently bound to
inactivated thymidylate
synthase making the drug
suicide inhibitor
 DHF can be reduced to THF by dihydrofolate reductase ,
inhibited by folate analogs such as methotrexate.
 Blocks reduction of DHF to THF by dihydrofolate reductase

 By decreasing the supply of THF, methotrexate

inhibits purine synthesis

 by preventing methylation of dUMP to dTMP, decrease the
availability of this essential component of DNA
 DNA synthesis inhibited and cell growth slowed ,treating
cancer
 Gene Expression & Enzyme Activity Both Are Regulated
 CAD represents the primary focus for regulation of pyrimidine
biosynthesis. Expression of the CAD gene is regulated at the
level both of transcription and of translation. At the level of
enzyme activity, the carbamoyl phosphate synthetase II (CPS)
activity of CAD is activated by PRPP and is feedback
inhibited by UTP. The effect of UTP is, however, abolished by
phosphorylation of serine 1406 of CAD.
 Purine and pyrimidine biosynthesis
parallel one another quantitatively,
that is, mole for mole, suggesting
coordinated control of their
biosynthesis. Several sites of cross-
regulation characterize the
pathways that lead to the
biosynthesis of purine and
pyrimidine nucleotides. PRPP
synthetase which forms a precursor
essential for both processes, is
feedback inhibited by both purine
and pyrimidine nucleotides, as is
the conversion of both pyrimidine
and purine nucleotides NDPs to
NTPs (Figure 33–10).
Acyclovir -a purine analog ,used to treat infections
of herpes simplex virus

• Inhibits the viral DNA polymerase

AZT -3 -azido-3 -deoxythymidine, a pyrimidine

analog ,used to treat human immunodeficiency
virus

• Inhibits the viral DNA polymerase.

 Allopurinol and the anticancer drug 5-fluorouracil are
alternate substrates for orotate
phosphoribosyltransferase.
 Both drugs are phosphoribosylated, and allopurinol is
converted to a nucleotide in which the ribosyl
phosphate is attached to N1 of the pyrimidine ring.
Pyrimidine ring opened and
degraded to highly water soluble
products
Hepatic β-ureidopropionase
catalyzes the formation of both β-
alanine and β-aminoisobutryrate
from their pyrimidine precursors.
End products of pyrimidine
catabolism :
➢ CO2
➢ NH3
➢ β-alanine from the degradation
of CMP and UMP
➢ β- aminoisobutyrate from TMP
degradation
 Excretion of β-aminoisobutyrate increases in leukemia and
severe x-ray radiation exposure due to increased destruction of
DNA.
 Many persons of Chinese or Japanese ancestry routinely excrete
β-aminoisobutyrate
 Disorders of beta alanine and beta aminoisobutyrate metabolism
arise from defects in enzymes of pyrimidine catabolism
 β hydroxybutyric aciduria : disorder due to total or partial
deficiency of the enzyme dihydropyrimidine dehydrogenase.
 Combined uraciluria-thyminuria: formation of β-alanine and β
aminoisobutyrate impaired . serious neurological complications .
 A nongenetic form triggered by the administration of the
anticancer drug 5-fluorouracil to patients with low levels of
dihydropyrimidine dehydrogenase
 Pyrimidine bases can be salvaged to nucleosides, which
are phosphorylated to nucleotides.
 mammalian cells reutilize few free pyrimidines
 Their high solubility makes pyrimidine salvage
insignificant
 Salvage reactions convert the pyrimidine
ribonucleosides uridine and cytidine and the
pyrimidine deoxyribonucleosides thymidine and
deoxycytidine to their respective nucleotides.
 Phosphoryltransferases (kinases) catalyze transfer of
the γ-phosphoryl group of ATP to the diphosphates of
the dNDPs 2′-deoxycytidine, 2′-deoxyguanosine, and
2′-deoxyadenosine, converting them to the
corresponding nucleoside triphosphates.
 NDP + ATP → NTP + ADP
 dNDP + ATP → dNTP + ADP
 No human enzyme catalyzes hydrolysis or
phosphorolysis of the pseudouridine (ψ) derived from
the degradation of RNA molecules.
 This unusual nucleotide excreted unchanged in the
urine of normal subject
 a very rare disorder, caused by a deficiency of one or both
activities of bifunctional enzyme , UMP synthase
 excessive excretion of orotic acid in urine
 Low activities one or both domains of UMP synthase
 Type1 orotic aciduria :deficiency of both orotate
phosphoribosyltransferase and orotidylate decarboxylase
 Type11 orotic aciduria : deficiency of orotidylate
decarboxylase
 Poor growth
 Megaloblastic anemia
 Inhibition of RNA and DNA synthesis and failure to thrive.
This can lead to mental and physical retardation
 Orotic aciduria in reye syndrome - a consequence of
the inability of severely damaged mitochondria to
utilize carbamoyl phosphate ,which becomes available
for cytosolic overproduction of orotic acid
 Reye's (Reye) syndrome is a rare but serious
condition that causes swelling in the liver and brain.
Reye's syndrome most often affects children and
teenagers recovering from a viral infection, most
commonly the flu or chickenpox
 Allopurinol an alternative substrate for orotate
phosphoribosyltransferase competes with orotic acid.
The resulting nucleotide product also inhibits
orotidylate decarboxylase resulting in orotic aciduria
and orotidinuria.
 6-azauridine competitively inhibits orotidylate
decarboxylase enhancing excretion of orotic acid and
orotidine
 Administration of uridine results in improvement of
anemia and decreased excretion of orotate
Purine Metabolism Pyrimidine Metabolism

Sources of the atoms in purine ring: Sources of the atoms in the pyrimidine ring
Aspartate, Glycine, :glutamine, CO2, and aspartate
Glutamine,tetrahydrofolate,CO2
In purine biosynthesis PRPP serves as a In pyrimidine biosynthesis PRPP
scaffold for assembly of the purine ring participates only subsequent to assembly of
the pyrimidine ring
energetically costly Less energy required

purine ring constructed on a preexisting pyrimidine ring synthesized before being

ribose 5- phosphate attached to ribose 5- phosphate

Both purine and pyrimidine synthesis Both purine and pyrimidine synthesis
require glutamine, aspartic acid, and PRPP require glutamine, aspartic acid, and PRPP
as essential precursors as essential precursors

purine ring, not cleaved in humans, Pyrimidine ring opened and degraded to
excreted as poorly soluble uric acid highly water soluble products :
CO2, NH3, β-alanine, β- aminoisobutyrate
Purine Metabolism Pyrimidine Metabolism

Intermediate : IMP Intermediate : UMP

Salvage is major pathway but can be de novo synthesis is major pathway

synthesized de novo

Methotrexate inhibits denovo synthesis Methotrexate inhibits dTMP synthesis