BMS204
Nucleotide Metabolism
Dr. Reem Arafa
Professor of Biomedical Sciences
Central Dogma of Biology
Nucleic Acids
Nucleic Acids Are Essential For Information Transfer in Cells
 Information encoded in a DNA molecule is transcribed via synthesis of an RNA
molecule
 The sequence of the RNA molecule is "read" and is translated into the sequence
of amino acids in a protein.
 Compounds containing C, N, O, and high amount of P.
 Was an acid compound found in nuclei therefore named nucleic acid
 1944 Oswald, Avery, MacLeod and McCarty demonstrated that DNA is the
molecular carrier of genetic information.
 1953 Watson and Crick proposed the double helix model for the structure of DNA
Nucleic acids as long polymers of nucleotides.
Nucleotides and Nucleic Acids
The amino acids sequence in a protein and nucleotide sequence in RNA is
specified by a nucleotide sequence in the cell’s DNA
Gene: segment of a DNA molecule that contains the information required for the
synthesis of a functional biological product whether a protein or RNA
The cell contains thousands of genes and DNA molecules
Nucleic Acids are polymers of nucleotides.
Deoxyribonucleic acid - DNA - Storage of genetic
information.
Ribonucleic acid - RNA - carriers of genetic
information and catalysis.
Nucleotide Structure: Nitrogenous base + ribose
+ phosphate
Nucleotides structure
The nucleotide has three characteristic components
 Nitrogenous base
 Pentose sugar
 Phosphate
Nitrogenous Bases
Nitrogenous base: derivatives of Purines and pyrimidines
DNA and RNA contain the same purine bases and the pyrimidine base
Cytosine but Thymine is found only in DNA and Uracil is found only in RNA
(A)
(C)
(G)
(U)
(T)
Pentoses of Nucleotides
•
•
•
•
D-ribose (in RNA)
2-deoxy-D-ribose (in DNA)
The difference is 2'-OH vs 2‘-H
This difference affects secondary
structure and stability
Bases are attached by β-N-glycosidic linkages to
carbon 1 of pentose sugar – (Nucleoside)
•
Base is linked via a β-N-glycosidic
bond
•
The carbon of the glycosidic bond
is anomeric
•
Named by adding -idine to the root
name of a pyrimidine or -osine to
the root name of a purine
•
Sugars make nucleosides more
water-soluble than free bases
Nucleosides
(1)
(9)
Nucleotides
• Phosphate ester of nucleosides
Nucleotides
The plane of the base is oriented perpendicular to
the plane of the pentose group
RNA contains Ribose while DNA contains 2'deoxy-D-Ribose
Ribonucleotides
Adenosine 5'-monophosphate, Adenylate, AMP
Guanosine 5'-monophosphate, Guanylate, GMP
Cytidine 5'-monophosphate, Cytidylate, CMP
Uridine 5'-monophosphate, Uridylate, UMP
Deoxyribonucleotides
Deoxythymidine 5‘-monophosphate,
Deoxythymidine, dTMP ……..
NMP=== Nucleoside mono phosphate.
Numbering of Ribose sugar is given 1’, 2’, …, 5’
Anti- conformation predominates in nucleic
acid polymers
• Conformation can be syn or anti
Unusual nucleotides
Modified nucleotides are found in some viral DNA and in Transfer RNA. These
modifications include methylation, hydroxymethylation, glycosylation, acetylation.
Other Functions of Nucleotides
• Nucleoside 5'-triphosphates are carriers of energy
• Bases serve as recognition units
• Cyclic nucleotides are signal molecules and regulators of cellular
metabolism and reproduction e.g. cyclic AMP
• Structural component of some coenzymes, e.g. CoA, FAD, NADH, NADPH
• ATP is central to energy metabolism
• GTP drives protein synthesis
• CTP drives lipid synthesis
• UTP drives carbohydrate metabolism
De novo synthesis of purine nucleotides
The atoms of purine ring are contributed by a number of compounds including amino
acids (aspartic acid, glycine and glutamine), CO2, and tetrahydrofolate. These
compounds donates N and C to constructed Ribose 5-phosphate.
6
5
1
2
4
3
7
8
9
N10Formyl tetrahydrofolate
The order in which ring atoms are added is:
9
Glut
4 5 7
Glycine
8
For
3
6
Glut
1
CO2
6
5
1 N
2
2
Asp
N 7
8
N
3
4
N
9
Ribose-P
For
De novo synthesis of purine nucleotides
* Synthesis of 5-phosphoribosyl-1-pyrophosphate (PRPP)
- Ribose 5-phosphate is synthesized from HMP (Hexose monophosphate
pathway)
- Ribonucleotides are first synthesized then reduced to deoxyribonucleotides
Inhibitors
Purines,
nucleosides
Activator
Pi
Ribose-5-phosphate
Ribose phosphate
pyrophosphokinase
ATP
AMP
Synthesis of 5-phospho-β-D-ribosylamine
•The amide group of the glutamine replaces pyrophosphate group attached to
PRPP, this reaction is mediated by Glutamine:phosphoribosyl
pyrophosphate amidotransferase.
•This enzyme is inhibited by
end product of this pathway
purine 5’-nucleotides AMP,
GMP and IMP.
• This reaction is the
committed step in purine
nucleotide synthesis
De novo synthesis of
purine nucleotides
Synthesis Inosine monophosphate
(IMP)
• IMP is the parent purine nucleotide
• Synthesis of IMP requires 4 ATP molecules
Inhibitors of Purine synthesis
• Specific inhibitors that inhibits the growth of
rapidly growing microorganisms e.g.
Sulfonamides
• Structural analogues for folic acid
(methotroxate)
* Conversion of IMP into AMP and GMP
- this reaction is energy-requiring pathway
Adenylosuccinate
synthetase
Feed back
inhibition
IMP dehydrogenase
Feed back
inhibition
Conversion of IMP into AMP and GMP
Conversion of Nucleoside momophosphate (NMP) to
nucleoside diphosphate (NDP) and triphosphate (NTP)
• NDP and NTP are synthesized from the corresponding NMP by Nucleoside
monophosphate kinases
• These kinases don't discriminate between ribose or deoxyribose in the substrate.
• ATP is the source of the transferred phosphate
examples
AMP + ATP  2 ADP  Adenylate kinase (highly active in the liver)
GMP + ATP  GDP + ADP  Guanylate kinase
• Nucleoside diphosphates and triphosphates are interconverted by nucleoside
diphosphate kinase
GDP + ATP  GTP + ADP
CDP + ATP  CTP + ADP
De novo synthesis of
purine nucleotides
Salvage Pathway for Purines
Salvage Pathway: Purines that results from the normal turnover of cellular nucleic
acids or obtained from the diet and not degraded can be reconverted into nucleoside
triphosphates and used by the body.
Two enzymes are involved:
 Adenine phosphoribosyl
transferase (APRT)
 Hypoxanthine-guanine
phosphoribosyl transferase (HGPRT)
Both enzymes utilize PRPP as the
source of ribose5-phosphate group
Degradation of Purine
nucleotides
Degradation of Purine nucleotides
 Purines are sequentially degraded into uric acids (in humans)
 Several steps are involved in this catabolic pathway
Degradation of Dietary nucleic acids in the small intestine
• Ribonucleases and deoxyriboncleases secreted in the pancreatic juice can
hydrolyze RNA and DNA into oligonucleotides.
• Oligonucleotides are further hydrolyzed by pancreatic phophodiesterases producing
a mixture of 3’-and 5’ mononucleotides
• A family of nucleotidases remove the phosphate group releasing nucleosides that
get absorbed from GIT
• Dietary nucleotides are not used to large extent in cells because they are converted
into uric acid in the small intestine and also used by the normal flora
Degradation of
Purine
nucleotides
Pyrimidine nucleotides synthesis
Sources of carbon atoms in pyrimidine rings
- Purine ring is synthesized on an existing ribose 5-phosphate
- Pyrimidine ring is synthesized then attached to ribose 5-phosphate donated by
PRPP
- the sources of carbon atoms in pyrimidine rings are Glutamine, CO2, and aspartic
acid
Aspartic Acid
CO2
Pyrimidine nucleotides synthesis
- Synthesis of carbamoyl phosphate
- The committed step of this pathway in mammalian cell is the synthesis of carbamoyl
phosphate from Glutamine and CO2
2 ATP + CO2 + Glutamine  Carbamoyl phosphate + 2ADP + Glutamate
- This reaction is mediated by Carbamoyl Phosphate Synthetase II (CPS II)
- CPS II is inhibited by UTP and activated by ATP and PRPP
- Carbamoyl phosphate is the precursor of Urea; the pyrimidine synthesis occurs in
the cytosol while the urea production occurs in the mitochondria by Carbamoyl
Phosphate Synthetase I (CPS I)
- CPS I uses ammonia as source of nitrogen
- CPS II uses the amide group of glutamine
- Glutamine is required in the synthesis of both Purines and Pyrimidine
Synthesis of carbamoyl phosphate
De Novo Pyrimidine Synthesis
De Novo Pyrimidine Synthesis
Degradation of pyrimidines
- Purines are not cleaved in human cell
- Pyrimidines rings can be opened and degraded to highly soluble structures,
such as -alanine and -aminoisobutyrate that can serve as precursors of
acetyl CoA and succinyl CoA
- Pyrimidine can be salvaged and converted into nucleotides by the enzyme
Pyrimidine phosphoribosyltransferase and it utilizes the PRPP
Conversion of Ribonucleotides to Deoxyribonucleotides
 2’-deoxyribonucleotides are synthesized from ribonucleoside diphosphatase
 Ribonucleotide reductase is multi-subunit enzyme (2B1 and 2B2 subunits) that
catalyzes the reduction of NDP (ADP, GDP, CDP, UDP) into dNDP (dADP, dGDP,
dCDP, dUDP)
 The immediate donors of hydrogen atoms needed for the reduction
are two–SH
groups of the enzyme itself
 The reduced form of the enzyme should be regenerated  The reducing agent is a
peptide coenzyme of ribonucleotide reductase called Thioredoxin
 The thioredoxin contain two cysteine residues that can be oxidized to reduce the
ribonucleotide reductase enzyme.
 The oxidized thioredoxin is reduced back by NADPH, this reaction is mediated by
Thioredoxine reductase
Regulation of Deoxyribonucleotides synthesis
 The regulation of this enzyme is complex
 Not only the activity is regulated but also substrate specificity
 The binding of dATP to an allosteric site called activity site inhibits the enzyme while
the binding of ATP to this site activate the enzyme.
 The binding of NTP to an allosteric site called substrate specificity will increase the
conversion of different NTP to their corresponding dNTP according to the need of the
cell
When ATP or dATP are bound to this
site  reduction of UDP, and CDP is
favored
When dTTP,or dGTP is bound  the
reduction of GDP, ADP is stimulated
Synthesis of deoxythymidine
monophosphate from dUMP
- dUMP is converted into dTMP by
thymidylate synthetase which utilizes
N5,N10 –methylene tetrahydrofolate as
the source of methyl group and 2
hydrogen  oxidation into
dihydrofolate.
- inhibitors of thymidylate synthetase
(5-flouro uracil) act as anti-tumor
- DHF can be reduced into THF by DHF
reductase, which can be inhibited by
Methotrexate  inhibits purine
synthesis and decrease the supply of
THF so prevents methylation of dUMP
to dTMP
The End