Download The Synthesis and Degradation of Nucleotides

The Synthesis and Degradation of Nucleotides
Objectives
I.
Activation of Ribose for Nucleotide Biosynthesis
A. Describe the synthesis of 5-phosphoribosyl-α1-pyrophosphate.
B. Describe the importance of this reaction.
C. Describe the allosteric control of this reaction.
II. Purine Biosynthesis
A. What molecules serve as the source of the atoms used in a the synthesis of a purine nucleotide?
B. Delineate the sequence of reactions (group additions) involved in the synthesis of a purine
nucleotide.
C. Detail the reactions leading from inosine-5´-monophosphate to adenosine-5´-monophosphate
and inosine-5´-monophosphate to guanosine-5´-monophosphate.
D. Note similarities among the reactions of the urea cycle and the reactions of purine biosynthesis.
E. Describe the control of purine biosynthesis.
1. The allosteric enzymes and molecules that control their activity.
III. Pyrimidine biosynthesis
A. What molecules serve as the source of the atoms used in a the synthesis of a pyrimidine
nucleotide?
B. Delineate the sequence of reactions / group additions involved in the synthesis of a pyrimidine
nucleotide.
C. Detail the reactions leading from uridine-5´-monophosphate to cytidine-5´-triphosphate.
D. Note similarities among the reactions of the urea cycle and the reactions of pyrimidine
biosynthesis.
E. Describe the control of pyrimidine biosynthesis.
1. The allosteric enzymes and molecules that control their activity
IV. Formation of Deoxynucleotides
A. Describe the enzymes and small molecules involved in the reduction of nucleotides to
deoxynucleotides.
B. Electron donor molecule
C. Delineate the flow of electrons through this “electron transport chain” to the ribose moiety.
D. Recount the control mechanisms of this enzyme.
V. Deoxythymidine Synthesis
A. Describe the enzymes and cosubstrates involved in this reaction.
B. What function(s) does N5, N10-methylenetetrahydrofolate serve during this reaction?
C. What is the medicinally important inhibitor of this reaction and what is its mechanism?
VI. Salvage Pathways
A. What are the purine salvage pathways?
B. Why are they important?
C. What enzymes are involved in these pathways?
D. Describe possible reason(s) for a lack of pyrimidine salvage enzymes.
VII. Purine Catabolism
A. Understand the general principles of the process.
1. What is the final product of this pathway?
2. What disease state is associated with excess purine catabolism and/or malfunctioning
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©Kevin R. Siebenlist, 2015
enzymes?
VIII. Pyrimidine Catabolism
A. Understand the general principles of the process.
1. What are the final products of cytidine or uridine catabolism?
2. What are the final products of thymidine catabolism?
a) How does the catabolism of thymidine interrelate with fatty acid and amino acid
catabolism?
IX. Integrate Nucleotide Metabolism with Carbohydrate Metabolism, Lipid Metabolism, & Amino
Acid Metabolism.
X. Ask yourself “What If Questions”
Nucleotide Biosynthesis
PURINE BIOSYNTHESIS starts with the formation of an activated ribose intermediate. Ribose is activated by
the enzyme Ribose-5-phosphate Pyrophosphokinase. An older name for the enzyme is 5-Phosphoribosyl-1pyrophosphate Synthase. This enzyme catalyzes the transfer of pyrophosphate from ATP to the anomeric
hydroxyl group, the hydroxyl group on carbon one of ribose-5-phosphate. The product of the reaction is 5phosphoribosyl-α1-pyrophosphate (PRPP) and AMP. PYRIMIDINE BIOSYNTHESIS and the PURINE SALVAGE
PATHWAYS (see below) also require PRPP, the activated ribose molecule.
ATP
AMP
Ribose-5-phosphate
Pyrophosphokinase
Ribose-5-phosphate Pyrophosphokinase is an allosteric enzyme. Its activity is inhibited when the cell
contains adequate amounts of the nucleotides.
Purine Biosynthesis
Purine nucleotide biosynthesis is a complex 10 step process. This
pathway will be very very briefly examined. The source of the
atoms that makeup the purine ring and the order in which they are
added to form the purine ring is necessary information
N1 is from Aspartate
C2 and C8 are donated by N10-Formyl-Tetrahydrofolate
N3 and N9 are donated by Glutamine
C4, C5 and N7 are from Glycine
C6 is from CO2 (HCO3–)
2
(6)
H
C
(1)
N
(7)
(5)
C
H
N
CH (8)
(2) HC
C
N
(3)
(4)
N
H
(9)
©Kevin R. Siebenlist, 2015
The pathway starts with Glutamine-PRPP Amidotransferase transferring the amide nitrogen of glutamine to
the anomeric carbon, C1 of 5-phosphoribosyl-1-pyrophosphate (PRPP). When the amide group is
transferred, the pyrophosphate on C1 is released and the configuration around the anomeric carbon switches
from α to β. The product is 5-phospho-β-D-ribosylamine. The transferred amine group is N9 of the purine
ring system.
O
O
P
O
O
H2
C
O
O
P
O
P
O
O
O
O
O
OH
OH
5-Phosphoribosyl-α1-pyrophosphate
H2O
Glutamine
Glutamine-PRPP
amidotransferase
(N9)
PPi
Glutamate
O
O
P
O
H2
C
NH2
O
O
OH
OH
5-Phospho-β-ribosylamine (PRA)
(2) - Glycine (C4, C5, and N7) is added to N9 of the growing purine.
(3) - A formyl group (C8) donated by N10-formyl-tetrahydrofolate is attached to the free amino group.
(4) - Glutamine now donates its amido group (N3) to the carbonyl carbon.
(5) - The five membered ring of the purine nucleus is closed by the enzyme AIR Synthetase.
(6) - CO2 (C6) is added to the growing purine ring system.
(7) - The amino group of aspartate is now linked to the just added carboxyl group.
(8) - The intermediated is cleaved releasing fumarate. The amino group of aspartate is left behind and it
becomes N1 of the purine.
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(9) - A formyl group (C2) from N10-formyl-tetrahydrofolate is now added.
(10) - In the last step the 6 membered ring of the purine nucleus is closed by the action of IMP
Cyclohydrolase. The product is INOSINE-5´-MONOPHOSPHATE (IMP).
IMP to AMP and GMP
Inosine-5´-monophosphate (IMP) is the precursor for Adenosine-5´-monophosphate (AMP) and
Guanosine-5´-monophosphate (GMP). IMP is converted to AMP in a two step pathway. In the first step
aspartate is added to the carbonyl group on C6 of IMP to form adenylosuccinate. This reaction is catalyzed
by Adenylosuccinate Synthetase. The energy released by the hydrolysis of GTP to GDP and PO4–3 is used
to drive the formation of the new chemical bond.
O
N
HN
O
O
P
N
N
O
CH2
O
O
GTP
Aspartate
OH
IMP
Adenylosuccinate
synthetase
IMP dehydrogenase
GDP + PO4–3
O
C
O
NADH
O
O
H
C
C
H2
C
O
N
N
O
O
P
N
N
O
CH2
N
HN
O
NH
O
H2O
NAD
OH
P
O
O
CH2
O
O
OH
O
OH
OH
Adenylosuccinate
H2O + ATP
Glutamine
Adenylosuccinate
lyase
GMP synthetase
P2O7–4 + AMP
Fumarate
Glutamate
NH2
O
N
N
O
P
N
N
O
CH2
N
HN
O H2N
O
O
O
P
O
N
N
CH2
O
O
OH
OH
Xanthosine monophosphate
(XMP)
O
O
N
N
H
OH
OH
OH
GMP
AMP
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The adenylosuccinate is now cleaved by the enzyme Adenylosuccinate Lyase to form AMP and fumarate.
The donation of an amino group by adding an aspartate and removing a fumarate is a repeating theme in
biosynthesis and it is one of the two ways in which amino/amide groups are transferred.
IMP is converted to GMP in two steps. First, H2O is added across the double bond between C2 and N3 of
the purine ring and the resulting hydroxyl group on C2 is oxidized to a carbonyl group. This reaction is
catalyzed by IMP Dehydrogenase. Xanthosine-5´-monophosphate (XMP) is the product.
Glutamine then donates its amide group to the newly formed carbonyl group to form GMP and glutamate.
During this reaction, catalyzed by GMP Synthetase, a molecule of ATP is hydrolyzed to AMP and two
phosphates to supply the energy for the formation of the new chemical bond. Glutamine donating its amide
group is the second method in which amino/amide groups are transferred.
Ribose-5-phosphate
Ribose-5-phosphate
Pyrophosphokinase
(–)
(–)
(–)
5-Phosphoribosyl-1-pyrophosphate
Glutamine-PRPP
Amidotransferase
(–)
(–)
(–)
5-Phosphoribosylamine
De novo pathway
(steps 2 - 10)
IMP
(–)
Adenylosuccinate
Synthetase
IMP Dehydrogenase
(–)
Adenylosuccinate
Xanthosine monophosphate
(XMP)
Adenylosuccinate
Lyase
(–)
GMP
Synthetase
AMP
GMP
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©Kevin R. Siebenlist, 2015
Control of Purine Biosynthesis
Purine nucleotide biosynthesis is an energy expensive pathway and as such it is tightly regulated. The cell
has no need to synthesize more purines than are absolutely necessary. This pathway is controlled at several
points.
IMP, AMP, and GMP are allosteric effectors for Ribose-5-phosphate Pyrophosphokinase. When their
concentrations are elevated, the activity of this enzyme is inhibited.
The Committed Step for purine biosynthesis is the reaction catalyzed by Glutamine-PRPP
Amidotransferase. This enzyme is allosterically inhibited by IMP, AMP, and GMP.
The pathways from IMP to AMP and from IMP to GMP are also allosterically controlled. AMP inhibits the
activity of Adenylosuccinate Synthetase. XMP and GMP inhibit the action of IMP Dehydrogenase.
There is cross talk between the pathways from IMP to AMP and from IMP to GMP assuring that AMP and
GMP are synthesized in balanced adequate amounts. The need for GTP in AMP synthesis and ATP in GMP
synthesis helps to maintain the balance between GMP and AMP concentrations in the cell. The IMP to AMP
pathway, the IMP to GMP pathway, and the control points of purine biosynthesis are important to know.
Pyrimidine Biosynthesis
Pyrimidine nucleotide biosynthesis is a much more straight forward process.
C2 of the pyrimidine ring comes from HCO3– (CO2), N3 comes from
glutamine, and the remainder of the pyrimidine molecule (N1, C4, C5 and
C6) comes from a molecule of Aspartate. The pyrimidine ring is
synthesized in four steps and then joined to PRPP to form the nucleoside-5´monophosphate. This is different from purine synthesis where the ring is
built step by step on ribose-5´-phosphate starting with PRPP.
(4)
H
C
(3) N
CH (5)
(2) HC
CH (6)
N
The pathway for the formation of pyrimidine nucleotides begins with the
(1)
formation of carbamoylphosphate. This reaction is catalyzed by
Carbamoylphosphate Synthetase II. This enzyme takes glutamine as the
ammonia donor, HCO3–, and 2 ATP molecules and catalyzes the formation of carbamoylphosphate. Other
products include glutamate, 2 ADP and a phosphate. This enzyme is different from Carbamoylphosphate
Synthetase I used for urea synthesis. Carbamoylphosphate Synthetase II is a cytosolic enzyme requiring
glutamine as the nitrogen donor, whereas Carbamoylphosphate Synthetase I is a mitochondrial enzyme that
utilizes ammonia.
Carbamoylphosphate is then condensed with a molecule of aspartate to form carbamoylaspartate. This
reaction is catalyzed by the enzyme Aspartate Transcarbamoylase.
Dihydroorotase catalyzes a dehydration reaction that results in closure of the pyrimidine ring.
Dihydroorotate is the product of this reaction. The first three enzymes of the pathway; Carbamoyl
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phosphate Synthetase II, Aspartate Transcarbamoylase, and Dihydroorotase; are contained on a single
multifunctional protein present in the cytoplasm.
O
O
O
C
H3N
C
C
HN
H
C
CH2
O
O
CH2
O
P
O
CH2
C
HCO3–
Carbamoyl phosphate
synthetase II*
HCO3–
2 ADP + PO4–3
Glutamate
O
OMP decarboxylase**
H2 O
O
O
P
O
O
Carbamoyl phosphate
O
C
O
HN
O
C
Aspartate
transcarbamoylase*
H 3N
C
C
C
N
O
O
O
H
P
O
CH2
O
O
C
O
O
Aspartate
O
CH
C
O
O
CH2
PO4–3
O
O
OH
OH
Uridine-5´-monophosphate
2 ATP + H2O
C
CH
N
O
O
NH2
Glutamine
H 2N
CH
OH
OH
Orotidine-5´-monophosphate
C
NH2
CH2
C
O
Pyrophosphate
Orotate
phosphoribosyltransferase**
CH
C
N
H
O
O
Carbamoyl aspartate
PRPP
O
C
HN
Dihydroorotase*
H 2O
CH
C
O
C
N
H
C
O
O
Orotate
O
C
HN
CH2
C
O
Dihydroorotate dehydrogenase
CH
N
H
C
O
L-Dihydroorotate
O
Q
QH2
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©Kevin R. Siebenlist, 2015
The dihydroorotate is oxidized to orotate by Dihydroorotate Dehydrogenase. In bacteria the enzyme is a
NAD-linked flavoprotein containing bound FAD, FMN and Fe-S centers. In eukaryotes the enzyme is
bound to the inner mitochondrial membrane. The electrons are immediately accepted by a quinone (a CoQ
like molecule) and then passed to the ET/OxPhos pathway for ATP generation.
Orotate is now coupled to PRPP to form Orotidine-5´-monophosphate (OMP). This reaction is catalyzed by
Orotate phosphoribosyl Transferase.
In the last step of the pathway OMP is decarboxylated by OMP Decarboxylase to form Uridine-5´monophosphate (UMP). The last two enzymes of the pathway; Orotate phosphoribosyl Transferase and
OMP Decarboxylase; are contained on a single multifunctional protein present in the cytoplasm.
Cytidine nucleotides are synthesized from UMP. However, before the uridine base can be converted to
cytidine the UMP must be phosphorylated to UTP.
ATP
ATP
ADP
ADP
The UTP is then converted to Cytidine-5´-triphosphate (CTP) by CTP Synthetase. This enzyme takes the
amide group from glutamine and attaches it to the carbonyl carbon, C4, of UTP. The hydrolysis of ATP
drives the reaction to completion. The other product is glutamate.
O
O
P
O
O
O
O
O
P
O
P
O
O
NH
O
N
OH
O
O
CTP Synthetase
O
OH
UTP
P
NH 2
O
N
O
O
CH 2
O
O
O
O
P
Gln
P
O
N
O
CH 2
O
OH
ATP
Glu
8
ADP
+
PO4–3
O
OH
CTP
©Kevin R. Siebenlist, 2015
Control of Pyrimidine Biosynthesis
Pyrimidine nucleotide biosynthesis is controlled at the step catalyzed by Carbamoyl phosphate Synthetase
II. This is an allosteric enzyme. PRPP and ATP activate the enzyme and UDP and UTP are allosteric
inhibitors of its activity.
Deoxyribonucleotides
The four ribonucleotides obtained from the biosynthesis pathways - AMP, GMP, UMP, and CTP are reduced
to the deoxyribonucleotides needed for DNA synthesis. Before they can be reduced to deoxyribonucleotides
they must all be converted to the nucleoside diphosphate forms. The addition or removal of phosphate from
the various nucleotides is accomplished by the Nucleoside Monophosphate Kinases or Nucleoside
Diphosphate Kinase (see below).
Adenylate
Kinase
AMP
ADP
ATP
Guanylate
Kinase
GMP
GDP
ATP
ADP
Nucleoside
Diphosphate
Kinase
Cytidylate
Kinase
UMP
UDP
ATP
ADP
CTP
CDP
ADP
ADP
ATP
The reduction reaction is catalyzed by the enzyme Ribonucleotide Reductase. This enzyme has associated
with it one of two possible small electron transport chains composed of two other proteins; one is an enzyme
and the other is a small disulfide containing protein. In the first system Thioredoxin Reductase and
Thioredoxin make up the electron transport chain and reduction of the ribose moiety of the nucleotides
precedes as follows:
HS
Reduced
Active
S
Oxidized
HS
HS
Thioredoxin
NADP
FADH2
Oxidized
S
S
O6P2O
CH2
S
FAD
HS
NADPH
+ H+
Reduced
3-
Reduced
SH
Base
O
OH
OH
Ribonucleotide
Reductase
3-
Oxidixed
Inactive
O6P2O
CH2
Base
O
SH
S
Thioredoxin Reductase
S
OH
H
H 2O
NADPH passes a pair of electrons to a FAD covalently linked to Thioredoxin Reductase to form NADP and
FADH2. The FADH2 then reduces a disulfide (–S–S–) group on Thioredoxin Reductase to a pair of thiol (–
SH) groups. FADH2 is oxidized to FAD. Thioredoxin Reductase passes the electrons to a disulfide (–S–S–)
on the protein Thioredoxin. The disulfide (–S–S–) on Thioredoxin is reduced to a pair of thiol (–SH) groups
and the thiols (–SH) on Thioredoxin Reductase are oxidized to a disulfide (–S–S–). Ribonucleotide
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©Kevin R. Siebenlist, 2015
Reductase picks up the elections from Thioredoxin, oxidizing the thiol (–SH) groups on Thioredoxin to a
disulfide (–S–S–) and reducing the disulfide (–S–S–) group on Ribonucleotide Reductase to a pair of thiol
(–SH) groups. Finally, the electrons are used to reduce the ribose moiety of the ribonucleotides to 2deoxyribose. During this reduction reaction the thiol (–SH) groups on Ribonucleotide Reductase are
oxidized to a disulfide (–S–S–).
In the second system Glutathione (a small molecule), Glutaredoxin Reductase (an enzyme), and
Glutaredoxin (a protein) make up the electron transport chain. The flow of electrons to reduce the ribose is
for the most part identical, except the NADPH reduces a molecule of Oxidized glutathione in the first step
of the electron pathway rather than reducing a molecule of FAD.
HS
Reduced
Active
S
Oxidized
HS
HS
Glutaredoxin
NADP
2 GSH
Oxidized
S
S
O6P2O
CH2
S
GSSG
HS
NADPH
+ H+
Reduced
3-
Reduced
SH
Base
O
OH
OH
Ribonucleotide
Reductase
3-
Oxidixed
Inactive
O6P2O
CH2
Base
O
SH
S
Glutaredoxin Reductase
S
OH
H
H 2O
Ribonucleotide Reductase has a unique control mechanism to assure that the deoxyribonucleotides are
synthesized in adequate and balanced amounts. This enzyme contains an Activity Site, a Specificity Site,
and the catalytic site. The Activity Site turns the enzyme “ON” or “OFF”; the Specificity Site controls
which nucleotide will be reduced; and the catalytic site performs the reduction.
When the Activity Site is occupied by ATP the enzyme is turned “ON”.
When the Activity Site is occupied by deoxy ATP the enzyme is turned “OFF”.
When the Specificity Site is occupied by ATP or deoxy ATP (dATP) then CDP or UDP is reduced.
When the Specificity Site is occupied by deoxyTTP (dTTP) then GDP is reduced.
When the Specificity Site is occupied by deoxyGTP (dGTP) then ADP is reduced.
The Specificity Site assures that the deoxyribonucleotides are synthesized in balanced and adequate
amounts. Remember that in DNA A pairs with T and G pairs with C.
1. When the concentration of ATP is high, the cell is energy rich, it has the energy to synthesize DNA
and divide. ATP binds to the activity site to turn the enzyme “ON”. ATP also binds to the specificity
site to stimulate the reduction of the pyrimidines, UDP and CDP. DeoxyUDP is the precursor of
deoxythymidine (dTTP), the base pair partner of dATP in DNA. High ATP concentrations stimulate
the synthesis of its partner in DNA and the partner of deoxyguanosine.
2. As dTTP concentrations build up it signals that the deoxy pyrimidines are present in adequate
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©Kevin R. Siebenlist, 2015
amounts for DNA replication. dTTP binds to the specificity site and stimulates the reduction of one
of the purines, GDP to dGDP.
3. As dGTP concentrations increase, it binds to the specificity site and stimulates the reduction of the
other purine. ADP is reduced to dADP.
4. As dATP concentrations increase they signal that all four deoxy nucleotide triphosphates are present
in adequate amounts for DNA replication. dATP replaces ATP in the activity site and the enzyme is
turned “OFF”.
S
ATP, dATP
Effectors that Determine
dTTP
Enzyme Specificity
dGTP
S
A
A
|
SH
|
SH
C
HS—
C
—SH
ATP, dATP - Effectors that
Determine Enzyme Activity
CDP, UDP
GDP
Substrates
ADP
Formation of DeoxyTMP from DeoxyUMP
Deoxyuridylate nucleotides are never incorporated into DNA. Two mechanisms assure that the
deoxyuridylate nucleotides are not incorporated into DNA. First, the enzyme Deoxyuridine Triphosphate
Diphosphohydrolase rapidly converts any deoxyUTP that is formed to deoxyUMP. Second, the deoxyUMP
is rapidly and quantitatively converted to deoxyTMP.
The conversion of deoxyUMP (dUMP) to deoxyTMP (dTMP) is catalyzed by the enzyme Thymidylate
Synthase. N5,N10-Methylene-Tetrahydrofolate (N5,N10-Methylene-TH4) serves two functions during the
course of this reaction. First it donates a one carbon fragment to the dUMP nucleotide. The one carbon
fragment donated by tetrahydrofolate is in the methylene (–CH2–) oxidation state. In the final product the
one carbon fragment is in the methyl (–CH3) oxidation state. During the second part of the reaction the
tetrahydrofolate molecule acts as a reducing agent. It donates a pair of hydrogen atoms to reduce the one
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©Kevin R. Siebenlist, 2015
carbon fragment from the methylene oxidation state to the methyl oxidation state. The enzyme Thymidylate
Synthase catalyzes the transfer of the one carbon methylene fragment from TH4 to C5 of uridine and it
simultaneously reduces the one carbon fragment to a methyl group. The products of this reaction are dTMP
and dihydrofolate.
O
HN
Deoxyuridylate
dUMP
O 3PO
O
O
CH 2
Thymidylate
dTMP
N
O 3PO
O
O
CH 2
OH
CH 3
HN
N
O
OH
Thymidylate
Synthase
N
H 2N
H
N
H
H 2N
H
H
HN
N
O
H 2C
N
H
H
HN
N
CH 2
N
H
N
C
H2
H
N
R
O
R
N5,N10-Methylenetetrahydrofolate
7,8-Dihydrofolate
NADPH + H+
Glycine
Dihydrofolate
Reductase
Serine
Hydroxymethyltransferase
Serine
NADP
H 2N
N
H
N
H
H
H
HN
N
H
C
H2
H
N
R
O
Tetrahydrofolate
Dihydrofolate is useless to the cell. It must be reduced to tetrahydrofolate if cellular metabolism is to be
maintained. The reduction process is catalyzed by the enzyme Dihydrofolate Reductase. NADPH and a
hydrogen ion (H+) donates the hydrogens and electrons necessary for the reaction. Once the TH4 is
reformed it accepts a one carbon fragment from serine or glycine and it is ready for the next cycle of
reactions.
The enzyme Dihydrofolate Reductase was the first target for cancer chemotherapeutic agents. dTMP
(dTTP) is needed for DNA replication, inhibiting the formation of dTTP would inhibit DNA replication.
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With DNA synthesis inhibited, cancer cells would cease to divide, and the tumor would stop growing. In
fact all rapidly dividing cells cease to multiply. The drug METHOTREXATE is a specific competitive inhibitor
of the enzyme Dihydrofolate Reductase. This enzyme inhibitor was the first cancer chemotherapeutic agent.
The side effects; hair loss, loss of appetite, etc.; are due to inhibition of normal cell division.
Tetrahydrofolate
Dihydrofolate
Methotrexate
Purine Salvage Pathways
The synthesis of purines is an energy expensive pathway and only a small amount of energy is recovered
during their degradation. To save energy the cell recycles as many of the purine nucleotides as possible
using the Purine Salvage Pathways.
During the digestion of food stuffs and cellular metabolism, the purine nucleotides are broken down to
phosphate, ribose (deoxyribose) and the bases adenine, guanine, and/or hypoxanthine. Hypoxanthine is the
purine base present on Inosine-5´-monophosphate, its the base on IMP.
The purine bases are salvaged by the action of two enzymes. Adenine phosphoribosyl Transferase couples
the adenine base to 5-phosphoribosyl-α1-pyrophosphate (PRPP) to form AMP. Hypoxanthine-Guanine
phosphoribosyl Transferase joins the hypoxanthine base to PRPP to form IMP and/or it attaches guanine to
PRPP to form GMP.
ÿÿ
Hypoxanthine + PRPP
Guanine + PRPP
ÿÿ
Hypoxanthine-Guanine
phosphoribosyl Transferase
Hypoxanthine-Guanine
phosphoribosyl Transferase
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IMP + pyrophosphate
GMP + pyrophosphate
©Kevin R. Siebenlist, 2015
Bacteria have a salvage pathway for the pyrimidine bases. Humans use Orotate phosphoribosyl Transferase
and OMP Decarboxylase to salvage pyrimidines.
Catabolism of Purines
NH2
N
N
AMP
O
N
HN
AMP
Deaminase
N
N
O
NH3
H2O
N
N
IMP
R5´P
GMP
R5´P
N
N
O
N
HN
Adenosine
Deaminase
N
N
PO4–3
O
NH3
H2O
Adenosine Ribose
N
Inosine
PO4–3
N
HN
N
H2N
Ribose
Guanosine
N
PO4–3
Purine
Nucleoside
Phosphorylase
Ribose-1phosphate
N
Ribose
Purine
Nucleoside
Phosphorylase
Ribose-1phosphate
O
Hypoxanthine
R5´P
5´-Nucleotidase
PO4–3
NH2
N
H2O
5´-Nucleotidase
PO4–3
N
H2N
H2O
H2O
N
HN
O
N
HN
N
H
N
H2O + O2
N
H2N
Guanase
NH3
O
N
HN
Xanthine
N
H
H2O
Xanthine
Oxidase
H2O2
N
HN
Guanine
O
N
H
N
H
H2O + O2
Xanthine
Oxidase
H2O2
O
O
C
H 2N
C
O
Urate
Oxidase
H
N
C
HC
N
H
Allantoin
N
HN
O
OH
N
H
CO2
1
/2 O2
+ H2O
O
14
N
H
N
H
Uric Acid
©Kevin R. Siebenlist, 2015
Excess purines and pyrimidines originating from ingested nucleotides or from routine turnover of cellular
nucleic acids are catabolized. Most intracellular purine bases are salvaged and pyrimidine salvage probably
occurs. Purine breakdown yields only waste products that must be excreted, whereas the pyrimidines yields
molecules that can enter metabolism for energy generation.
During the catabolic process of AMP can be converted to IMP by AMP Deaminase and then the IMP is
converted to inosine (a nucleoside) by the enzyme 5´-Nucleotidase, or AMP is first dephosphorylated to
adenosine (a nucleoside) by 5´-Nucleotidase and then the adenosine is converted to inosine by Adenosine
Deaminase. The net result of these two pairs of reactions is the conversion of AMP (a nucleotide) to inosine
(a nucleoside). The inosine is then phosphorolytically cleaved, phosphate is added across the N-glycosidic
bond, to yield the base hypoxanthine and ribose-1-phosphate by the enzyme Purine Nucleoside
Phosphorylase.
Hypoxanthine is converted to xanthine by the action of the enzyme Xanthine Oxidase. This enzyme uses
molecular oxygen (O2) to oxidize hypoxanthine to xanthine and hydrogen peroxide (H2O2). Hydrogen
peroxide is a very destructive compound to have within the cell. It is rapidly and quantitatively destroyed
by the enzyme Catalase. Xanthine Oxidase resides in lysosomes and peroxisomes.
Xanthine is then converted to uric acid, the final excretory product in mammals, by a second reaction
catalyzed by Xanthine Oxidase.
GMP catabolism is similar. GMP is first dephosphorylated to guanosine (a nucleoside) by the action of 5´Nucleotidase. The guanine base is then released from the nucleoside by Purine Nucleoside Phosphorylase.
The guanine base is converted to xanthine by the enzyme Guanase. Once formed, xanthine is converted to
uric acid by the action of Xanthine Oxidase.
In Mammals the uric acid is usually oxidized to Allantoin by Urate Oxidase and the allantoin is the major
secretory product.
Pyrimidine Catabolism
The pyrimidine nucleotides are converted to their respective nucleosides by the action of 5´-Nucleotidase.
Cytidine (nucleoside) is converted to uridine (nucleoside) by the action of Cytidine Deaminase.
Ribose is removed from uridine by the enzyme Uridine Phosphorylase to release the free base uracil, and it
is removed from thymidine by the action of Thymidine Phosphorylase to release the free base thymine.
The enzyme Dihydrouracil Dehydrogenase reduces the bases uracil and thymine to dihydrouracil and
dihydrothymine, respectively.
These two compounds are then acted upon by the enzyme. Dihydropyrimidinase to form ureidopropionate
or ureidoisobutyrate.
15
©Kevin R. Siebenlist, 2015
NH2
CMP
N
O
O
O
HN
N
UMP
N
O
R5´P
dR5´P
O
O
N
O
HN
N
N
O
Cytidine
dTMP
R5´P
NH2
CH3
HN
O
Uridine
N
Ribose
CH3
HN
dThymidine
N
O
Ribose
Deoxyribose
O
O
C
HN
CH
C
CH
O
Uracil
N
H
O
O
C
C
HN
CH2
HN
C
CH2
C
Dihydrouracil
O
N
H
Thymine
CH
O
C
C
N
H
CH3
C
HN
CH
O
N
H
CH3
CH2
Dihydrothymine
O
O
H2
C
C
H2N
N
H
CH3
C
C
H2
H2N
O
H
N
C
O
Ureidopropionate
CH
C
H2
O
C
O
Ureidoisobutrate
16
©Kevin R. Siebenlist, 2015
O
CH3
O
H2
C
C
H2N
N
H
H
N
H2N
C
C
H2
CH
C
H2
C
O
O
Ureidopropionate
O
C
O
Ureidoisobutrate
ÿÿ
ÿÿ
CH3
O
H2
C
H3N
C
H3N
C
H2
CH
O
C
C
H2
O
O
-Alanine
-Aminoisobutyrate
O
O
C
H
O
C
C
H2
O
C
O
H
C
CH
O
CH3
Malonic Semialdehyde
Methylmalonic Semialdehyde
ÿÿ
ÿÿ
O
CoA
O
C
S
O
C
C
H2
CoA
O
O
C
S
C
CH
O
CH3
Malonyl-CoA
D-Methylmalonyl-CoA
The enzyme Ureidopropionase hydrolytically removes NH4+ and HCO3– from these compounds to form βalanine (from uracil) and β-aminoisobutyrate (from thymine).
An Aminotransferase (Transaminase) converts β-alanine into malonic semialdehyde and converts βaminoisobutyrate into methylmalonic semialdehyde.
A Dehydrogenase Complex oxidizes malonic semialdehyde and couples it to Coenzyme A to form malonyl17
©Kevin R. Siebenlist, 2015
CoA. The malonyl-CoA can enter fatty acid biosynthesis or more likely it is decarboxylated by MalonylCoA Decarboxylase to acetyl-CoA. and the acetyl-CoA oxidized for energy (ATP).
The same or a similar Dehydrogenase Complex oxidizes methylmalonic semialdehyde and couples it to
CoA forming D-methylmalonyl-CoA. D-methylmalonyl-CoA is an intermediate in the metabolism of odd
chain length fatty acids and the amino acids, Met, Val, Thr, and Ile. D-methylmalonyl-CoA is ultimately
converted to succinyl-CoA as described previously.
Deoxyribose Metabolism
HOCH 2
O
Base O
O
HOCH 2
P
O
O
OH
Nucleoside
O
O
O
P
O
CH2
O
OH
PO43–
OH
deoxyribose-1-phosphate
Base
OH
O
deoxyribose-5-phosphate
O
C
H 3C
O
O
C
S
CoA
H 3C
acetyl-CoA
C
O
H 3C
acetate
AMP
+
P4O7–4
CoA
+
ATP
H
ethanal
NADH
NAD
H
O
C
CH
+
glyceraldehyde3-phosphate
OH
H 2C
O
O
P
O
O
After the base is phosphorylytically released from the deoxyribose by Nucleoside Phosphorylase or
Thymidine Phosphorylase the phosphate is moved from C-1 to C-5 by Phosphopentose Mutase to form
deoxyribose-5-phosphate. The deoxyribose-5-phosphate is cleaved to ethanal and glyceraldehyde-3phosphate by 2-Deoxyribose-5-phosphate Aldolase. Carbon 1 & 2 becomes the ethanal and 3, 4, &5
become glyceraldehyde-3-phosphate. The glyceraldehyde-3-phosphate enters glycolysis or gluconeogenesis
depending upon the tissue and blood glucose levels. Ethanal is oxidized to acetate by Aldehyde
Dehydrogenase and then the acetate is coupled to Coenzyme A by Acetyl-CoA Synthetase. Acetyl-CoA
enters any of the pathways that utilizes Acetyl-CoA, most likely the TCA cycle.
18
©Kevin R. Siebenlist, 2015