2. Overview
• Structural aspects and history
• de novo synthesis of Pyrimidine bases.
• Regulation of synthesis.
• Synthesis of deoxy pyrimidine bases.
• Catabolism of pyrimidine bases..
• Disorder of pyrimidine metabolism.
4. Structure of Pyrimidine bases
• Pyrimidine bases: CUT
• Pyrimidine nucleotides are
- CMP
- UMP
- TMP
5. History
Adolf Pinner
first
proposed the
name
“pyrimidine”
in 1885.
Discovered by
Albrecht Kossel and
Albert Neumann in
1894, it was
hydrolyzed from calf
thymus.
unstable and can
change to uracil
(spontaneous
deamination).
Demethylated form
of thymine.
Discovered in 1900
by Alberto Ascoli.
Planar, unsaturated
compound, absorbs
light.
Only in RNA.
First isolated in
1893 by Albrecht
Kossel and Albert
Neumann from
calf thymus glands,
hence its name.
May be derived by
methylation of uracil
at the 5th carbon.
Only in DNA.
7. Synthesis: de Novo or Salvage
• de Novo Synthesis: nucleotide
bases are assembled from
simpler compounds.
• Salvage Pathway : preformed
bases are recovered and
reconnected to ribose unit.
8. • Denovo pathways : synthesis
from aspartate, glutamine,
ribose 5-phosphate, CO2 and
ATP.
• Different from purine de novo
synthesis, first pyrimidine ring is
assembled and linked to ribose
phosphate.
• Ammonia used here is usually
produced from hydrolysis of
sidechain of glutamine.
9. 1. Carbamoyl phosphate synthetase
• First Step: Activation
• Bicarbonate and ammonia with 2
ATP gives carbomyl phosphate in
multistep, catalyzed by carbomyl
phosphate synthetase II (CPSase II).
10. Structure of CPSase II
• 2 chain with 3 catalytic
domains:
• Compartmentation provides 2
independent pools of carbamoyl
phosphate.
11. 2. Aspartate transcarbamoylase
• Addition of aspartate.
• Committed step in pyrimidine biosynthesis.
• Inhibited by CTP (end product),
• Activated by ATP in bacteria.
12. 3. Dihydro-orotase: Cyclization
• Formation of dihydro-oratate
• Closure of ring with loss of water.
• First 3 enzymes are multifunctional polypeptides (CAD).
• Ensures efficient channeling for high energy carbamoyl
phosphate to pyrimidine biosynthesis.
13. 4. Dihydroorotate dehydrogenase
• Dehydrogenase cause formation
of double bond.
• Occurs in inner mitochondrial
membrane
• The ultimate electron
acceptor is NAD+
14. 5,6. UMP Synthase
• Orotate phosphoribosyl transferase and OMP decarboxylase
(bifunctional enzyme) are separate domains of a single
polypeptide—UMP synthase.
• Addition of ribose 5-phosphate from PRPP to form
orotidylate.
• Removal of carboxylic group from orotidylate form uridylate
(UMP)
15.
16. 7. Formation of CTP and TMP
THF
Serine
hydroxylmeth
yl transferase
Glycine
Serine
Dihydrofolat
e reductase
Methotrexate
Trimethoprim
5-Fluoro uracil
Hydroxy urea
17. Salvage of Pyrimidines
• Few Pyrimidine
bases are salvaged
in human cells.
• Salvaged by
Nucleoside
kinases.
• Salvage pathway
is used for
treatment of
Hereditary orotic
aciduria.
18. Regulation of Synthesis
- CPSase II: inhibited by UTP and
purine nucleotides, activated by
PRPP.
- Aspartate transcarbamoylase is
inhibited by CTP but activated by
ATP in bacteria
19. Ribonucleotide Reductase
• Key enzyme for evolution
mediates the synthesis of
deoxyribonucleotides.
• Substrates for DNA synthesis
RNA DNA
20. Coordinate regulation of
Purine and pyrimidine synthesis
• For each mol of deoxy purine nucleotide, deoxy pyrimidine
nucleotide needs to be synthesized, which is regulated by
ribonucleotide reductase allosteric regulation mechanism.
21. Biosynthesis of Thymidylate
• DNA contains thymine
rather than uracil and the
de novo pathway to
thymine involves only
deoxyribonu- cleotides.
• Immediate precursor of
thymidylate (dTMP) is
dUMP.
• Pathway to dUMP begins
with formation of dUTP,
either by deamination of
dCTP or by
phosphorylation of
dUDP.
22. Conversion of dUMP to dTMP is
catalyzed by thymidylate synthase.
- A one-carbon unit at the
hydroxymethyl oxidation level is
transferred from N5, N10-
Methylenetetrahydrofolate to
dUMP, then reduced to a methyl
group.
- Reduction occurs at the expense
of oxidation of tetrahydrofolate
to dihydrofolate, which is
unusual in tetrahydrofolate-
requiring reactions.
- Dihydrofolate is reduced to
tetrahydrofolate by
dihydrofolate reductase
23. Mono, di and triphosphates are
interconvertible
• Active forms are NDP and NTPs.
• Monophosphate to diphosphate by nucleoside
monophosphate kinases with ATP as phosphoryl donor (only
base specific).
UMP + ATP UMP Kinase UDP + ADP
• Diphosphate and Triphosphate are interconverted by
nucleoside diphosphate kinase (nonspecific).
XDP + YTP nucleoside diphosphate kinase XTP + YDP
24. Chemotherapeutic Agents Target Enzymes
• Useful targets for pharmaceutical agents are thymidylate
synthase and dihydrofolate reductase, enzymes that provide
only cellular pathway for thymine synthesis.
• Fluorouracil is an inhibitor that acts on thymidylate synthase,
chemotherapeutic agent.
• Fluorouracil itself is not the enzyme inhibitor.
• In the cell, salvage pathways converts it to the deoxynucleoside
monophosphate FdUMP, which binds to and inactivates the
enzyme
25. • Methotrexate is an inhibitor of dihydrofolate reductase.
• This folate analog acts as a competitive inhibitor; the
enzyme binds methotrexate with about 100 times higher
affinity than dihydrofolate.
• Aminopterin is a related compound that acts similarly.
• Trimethoprim, an antibiotic binds to bacterial dihydrofolate
reductase nearly 100,000 times better than to the
mammalian enzyme.
26. Catabolism of Pyrimidine
• Unlike purine, not cleaved, pyrimidine
ring is opened and degraded to soluble
products like β-alanine,
β-aminisobutyrate, NH3 and CO2.
27. • β-alanine excreted or
converted to carnosine or
anserine.
• β-aminisobutyrate is
excreted in urine or
transaminate to from
methylmalonyl semialdehyde
and finally to succinyl-CoA.
• Excretion of β
aminoisobutyrate increase in
leukemia.
Fig
:
28. Disorders of Pyrimindine metabolism
• Since the product of pyrimidine catabolism are highly
water soluble, overproduction of pyrimidine catabolites is
rarely associated with clinically significant abnormalities.
• Disorder related to Pyrimidine Synthesis
- Orotic aciduria
29. Disorders of Pyrimindine metabolism
Orotic Aciduria: Hereditary
autosomal recessive disorder.
• Cause:
- Deficiency in UMP
synthase (Orotate Phosphoribosyl
Transferase and Orotidylate
Decarboxylase).
- Secondary to blockage of
Urea cycle by Ornithine
transcarbamylase deficiency
distinguished by decrease in
urea with hyperammonemia.
• .
30. Two Types
Type I – There is deficiency of both enzymes orotate
phosphoribosyltransferrase and OMP decarboxylase
Type II – Only OMP decarboxylase is deficient
https://www.amjmed.com/article/0002-9343(73)90260-X/fulltext
31. Clinical features and treatment
• There is retarded growth and megaloblastic anemia
• Crystals excreted in urine
• Crystalluria may cause UTI
Treatment:
Oral administration of uridine bypass metabolic block and
provide source of pyrimidines
32. Reye’s Syndrome or Secondary Orotic Aciduria
• This presents similar to hereditary orotic aciduria and is not
due to defective pyrimidine nucleotide synthesis but defective
mitochondria.
• Ornithine transcarbomylase of urea cycle is defective so
carbomyl phosphate diffuses out into cytosol to lead mild
orotic aciduria
• It is associated with high blood ammonia and glutamine levels,
• In this condition ammonia metabolism is major concern. Orotic
aciduria is of secondary importance.
34. Lab Assessment
• Orotic acid can be converted to barbituric acid by bromination and
reduction in presence of ascorbic acid.
• Barbituric acid is coupled with p-dimethylaminobenzaldehyde to form
orange product.
• 5-( p-dimethylaminobenzylidene) barbituric acid, which absorbs light
at 480 nm.
• Orotic acid are measured by this method unless the metabolites are
separated chromatographically before analysis
Salerno C, Crifò C. Diagnostic value of urinary orotic acid levels: applicable separation methods. J Chromatogr B Analyt Technol Biomed Life Sci. 2002
Dec 5;781(1-2):57-71. doi: 10.1016/s1570-0232(02)00533-0. PMID: 12450653.
35. Drugs may precipitate Orotic aciduria
• Allopurinol: alternative substrate for orotate
phosphoribosyltransferase competes with orotic acid,
resulting nucleotide product inhibit orotidylate
decarboxylase resulting in orotic aciduria.
• 6-Azauridine: by conversion to 6-azauridylate also
competitively inhibits orotidylate decarboxylase,
resulting orotic aciduria.
36. References
• Lehninger Principles of Biochemistry
• Harper’s Illustrated Biochemistry
• Lippincott’s illustrated biochemistry
• Biochemistry Stryer
• https://www.amjmed.com/article/0002-9343(73)90260-
X/fulltext
• https://sohebbasharat.wordpress.com/2-16/02/20/orotic-
aciduria
• Salerno C, Crifò C. Diagnostic value of urinary orotic acid levels:
applicable separation methods. J Chromatogr B Analyt Technol
Biomed Life Sci. 2002 Dec 5;781(1-2):57-71. doi: 10.1016/s1570-
0232(02)00533-0. PMID: 12450653.
Nucleoside: Nitrogenous base + Pentose sugar
Nucleotide: Nucleoside + Phosphate
Cytidine monophosphate
Uridine monophosphate
Thimidine monophosphate
-The specific role of the carbamoyl phosphate synthetase I enzyme is to control the first step of the urea cycle, a reaction in which excess nitrogen compounds are incorporated into the cycle to be processed.
Deamination is the removal of an amino group from a molecule.
The Pyrimidine ring is assembled de Novo or Recovered by salvage pathways.
The framework of pyrimidine base is assembled first and then attached to ribose whereas in purine base is synthesized piece by piece directly onto ribose based structure
Phosphoribosyl pyrophosphate (PRPP) ribose 5-phosphate + ATP → PRPP + AMP. Ribose sugar from HMP shunt
deoxy pyrimidine nucleotide needs to be synthesized
The specific role of the carbamoyl phosphate synthetase I enzyme is to control the first step of the urea cycle, a reaction in which excess nitrogen compounds are incorporated into the cycle to be processed.
Carbamoyl phosphate synthetase is isolated from Escherichia coli as a heterodimeric protein.
The smaller of the two subunits catalyzes the hydrolysis of glutamine to glutamate and ammonia. The larger subunit catalyzes the formation of carbamoyl phosphate using 2 mol of ATP, bicarbonate, and ammonia.
multi-subunit protein complex composed of 12 subunits
The particular arrangement of catalytic and regulatory subunits in this enzyme affords the complex with strongly allosteric behaviour with respect to its substrates.
ATCase does not follow Michaelis–Menten kinetics. Instead, it lies between its low-activity, low-affinity "tense" and its high-activity, high-affinity "relaxed" states
The committed step is the one after which the substrate has only one way to go.
Carbamoyl phosphate condenses with aspartate to form carbomyl aspartate.
dihydro-oratase catalyses the ring closure by loss of water molecule
The three enzymes are the domain or functional unit of the same protein.
This reaction is catalyzed by dihydroorotate dehydrogenase
Oxidized by NAD+ and getting itself reduced to the NADH
Orotic acid is converted to Orotodine MonoPhosphate by enzyme orotate phosphoribosyl transferase and PRPP is the donor of ribose.
Then OMP is decarboxylated to UMP by enzyme orotidylic acid decarboxylase.
PRPP is derived from the first reaction of purine synthesis faciliated by the enzyme PRPP synthase
Hydroxy Urea (antimetabolites): inhibition of the enzyme ribonucleotide reductase by scavenging tyrosyl free radicals. Sickle cell dz, myloproliferative dz, Psoriasis, CML etc
Fluorouracil, an anticancer drug, is converted in vivo into fluorodeoxyuridylate(F-dUMP). This irreversibly inhibits thymidylate synthase after
Methotrexate: Anticancer and anti rheumatic arthritis: prevent rapid turnover of immune cells.
Trimethoprim- sulfamethoxazole (in bacteria): prokaryotes: UTI and mid ear infection :binds bacterial DFR 100,000X than human.
The major folate present in mammalian cells is 5-methyl-tetrahydrofolate (5-MTHF), which provides methyl groups to the S-adenosylmethionine (SAM) cycle through the conversion of homocysteine (Hcy) to methionine
Pyrimidines can be synthesized or reprocessed or salvaged from old pyrimidines.
So the breakdown of DNA can lead in formation of cytosine and Thymine. Now for uracil co factor is ribose 1 phosphate.
Uracil can come from RNA also.
Ribose 1 phosphate and deoxyribose 1 phosphate are less amount or abundant in the cell and only less amount of pyrimidines is processed.
For a given enzyme concentration, the value of enzyme reaction increases or decreases with increasing or decreasing substrate concentration
Ribose sugar needs to be converted to deoxyribose sugar to make deoxy ribose phosphate for DNA synthesis which is done by the single enzyme ribonucleotide reductase.
ribonucleotide reductase is the enzyme that ensures there is the balance in the synthesis of all deoxyribonucleotide triphosphate.
Faciliated by formation of water molecule . Hydroxyl group of C2 carbon is converted to water facilitated by thiol group containing Thioredoxin and first of all deoxyribose diphosphate is made.
For balance synthesis of deoxynucleotides the enzymes should be highly regulated.
So positive allosteric regulator is ATP and negative modulator is dATP. When ATP gets bind to the allosteric site first of all CDP gets bind to the active site and converted as dCDP then converted to dCTP then in second dUDP gets bind to allosteric site converting to TTP. TTP now increases and then replaces dATP in the allosteric binding site then GDP will bind to the active site converting to dGDP and to dGTP then dGDP replaces dTTP in the allosteric site and ADP binds to the active site converting to dADP and dATP.
Since dATP is a negative modulator of the enzyme so after it gets bind to the allosteric site the process of formation stops.
Hydroxymethyl group- CH2OH.
Dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase— a regeneration that is essential for the many processes that require tetrahydrofolate.
In plants and at least one protist, thymidylate synthase and dihydrofolate reductase reside on a single bifunctional protein.
The major folate present in mammalian cells is 5-methyl-tetrahydrofolate (5-MTHF), which provides methyl groups to the S-adenosylmethionine (SAM) cycle through the conversion of homocysteine (Hcy) to methionine
It is used to treat certain urinary and middle ear bacterial infections.
Aminopterin is an amino derivative of folic acid, which was once used as an antineoplastic agent in the treatment of pediatric leukemia
Parasitic protists, such as the trypanosomes that cause African sleeping sickness (African trypanosomiasis), lack pathways for de novo nucleotide biosynthesis and are particularly sensitive to agents that interfere with their scavenging of nucleotides from the surrounding environment using salvage pathways.
Allopurinol and several similar purine analogs have shown promise for the treatment of African trypanosomiasis and related afflictions.
α-DifluoromethylOrnithine (DFMO)
Inhibitor of Ornithine Decarboxylase
Nucleotidase is the enzyme removes the phosphate group.
Phosphorylase enzyme removes the sugar
Deaminase (incorporation of water molecule) , Reductase
Hydrolase (both).
CO2 and NH3 comes out ..
In human beings, beta-amino-iso-butyrate is trans-aminated to methylmalonate semialdehyde which is further converted to succinyl-CoA
Carnosine and anserine are strong antioxidants,
A defect in the UMP synthase results in the excretion of orotic acid in urine.
Feeding cytidine and Uridine (Cytidine is a drug used to manage neuropsychiatric deficits associated with cerebrovascular diseases in combination with uridine)
Uridine and choline work synergistically with DHA(Docosahexaenoic acid)omega 3 fatt acid to increase phosphatidylcholine formation
animal muscle is naturally rich in ATP, meat (pork, beef, chicken), fish and shrimps are excellent sources of purine nucleotides; 2) Baker yeasts are naturally rich in RNA and yeast extracts are excellent sources of both purine and pyrimidine nucleotides
Ornithine transcarbomylase
Since histidine do not form color so if there is presence of histidine amino acid interference, bromination is required
6-Azauridine is a synthetic triazine analogue of uridine with antimetabolite activity