Download Nucleotide Catabolism

Nucleotide Catabolism
April 26,2003
Bryant Miles
Ribonucleotide Reductase produces dADP,dGDP, dCDP and dUDP. These
diphosphodeoxyribonucleosides are phosphorylated by ATP to generate the dNTPs. The enzyme is the
same nucleoside diphosphate kinase that phosphorylates the ribonucleotides.
dNDP + ATP
dNTP + ADP
Nucleoside diphosphate kinase
NDP + ATP
NTP + ADP
Nucleoside diphosphate kinase
I. Thymine Biosynthesis
Ribonucleotide reductase converts CDP to dCDP and UDP to dUDP. Nucloeside diphosphate kinase
phosphorylates dUDP to form dUTP. dUTP is immediately hydrolyzed into dUMP and pyrophosphate by
dUTP phosphosphorylase. This reaction is important because the concentration of dUTP must be
minimized to prevent the incorporation of uracil into DNA. DNA polymerases do not discriminate
between dUTP and dTTP as substrates.
dUTP
dUMP + PPi
dUTP Phosphorylase.
Deoxyuridine monophosphate is methylated to form deoxythymidine monophosphate by thymidylate
synthase. Thymidylate synthase methylates dUMP using N5,N10-methylene-THF as the methyl donor.
O
Note that the methylene carried by the THF
H
has the same oxidation state as a
HN
H2N
N
N
hydroxymethyl group. This methylene is
CH2
H
transferred to the uracil and then reduced to
O
N
O
N
a methyl group. The reduction occurs by
H
CH2
N
+
-O P O
O
transferring a hydride from tetrahydrofolate
H
H
OH2C
O
N
which produces dihydrofolate.
H
H
OH
H
R
5 10
dUMP
Dihydrofolate is reduced back to
N ,N -Methylene-THF
tetrahydrofolate by dihydrofolate reductase.
Dihydrofolate reductase uses NADPH as
O
H
the reductant.
CH2
HN
O
O
-O
P
O
H
OdTMP
H
OH
N
O
H
H
H
N
CH2
N
+
H
N
H2N
N
H
O
Dihydrofolate
CH2
HN
R
This is an interesting reaction. Let’s check out the mechanism.
O
HN
O
O
-O
P
O
N
S
OH
dUMP
H
H
OH
H
O
H
N5,N10-Methylene-THF
P
N
O
-
H2C
O
-O
P
O
CH2
S
H
H
H
OH
H
N
+
B
O
-O
N
-O
P
O
O
C
H
2
HN
N
S
ENZ
H
O
H
H
H
H
OH
H
H
:B
dTMP
R
H
O
O
H
CH2
O
P
O-
H
R
CH2
O
O
H
N
CH2
HN
Dihydrofolate
HN
H
ENZ
N
O
ENZ
N
O
H
R
CH2
HN
H
H
N
N
O
OdUMP
N
OH
H2N
R
HN
H
H
N
H
H
CH2
H
S
O
OH
CH2
N
H
N
N
HN
CH2
O
O
H
N
N
H
CH2
O
HN
O
O
N
H
-O
O
CH2
:B
H2N
H2N
N
ENZ
H
O
H
N
N
H2N
N
S
:B
ENZ
O
OH
H
H
OH
H
H
1. Thymidylate synthase has a nucleophilic cysteine residue Cys-146 that attacks the C6 carbon of
dUMP to form a covalent intermediate.
2. The attack of the cysteine generates an enolate ion that attacks the –CH2- carried by N5,N10methylene-THF to form an enzyme-dUMP-THF ternary complex.
3. An enzyme base abstracts the proton from the C5 position of dUMP to eliminate the THF
cofactor.
4. A hydride is transferred from C6 of THF to the methylene group on the pyrimidine to form dTMP
and dihydrofolate.
Tetrahydrofolate is regenerated from dihydrofolate by NADPH. This reduction is catalyzed by
dihyrofolate reductase. The N5,N10-methylene-THF is regenerated by serine hydroxymethyl transferase
converting serine into glycine.
II. Anti-Tumor Reagents.
Cancer cells are rapidly proliferating cells. Normal mammalian cells grow and proliferate slowly.
Because cancer cells are rapidly growing and proliferating they rapidly consume nucleotides for the
synthesis of RNA and DNA. The result is cancer cells are more susceptible to inhibitors of nucleotide
synthesis than normal cells. When studying pyrimidine and purine nucleotide biosynthesis, you soon
realize than glutamine is the major source of nitrogen for nucleotide biosynthesis. These enzymes that
use glutamine as the nitrogen source all contain a homologous glutamine amidotransferase domain. Let’s
list these enzymes.
1. CPSII
2. CTP synthetase
3. Amidophosphoribosyl transferase
4. FGAM synthetase
5. GMP synthetase
Rapidly proliferating cancer cells are voracious consumers of
glutamine. They require glutamine for pyrimidine and purine synthesis.
Inhibitors of these glutamine amidotransferases have potential as
chemotherapeutic agents. Shown to the left are 2 potent inhibitors of
glutamine amido transferases, azaserine and acivicin. Both these
inhibitors irreversibly inactivate the GAT subunits.
The other targets for pharmaceutical reagents are thymidylate synthase
and dihydrofolate reductase. One inhibitor of thymidylate synthase is
fluorouracil. Fluorouracil itself does not inhibit anything, but in cells
fluorouracil is a substrate for pyrimidine phosphoribosyltransferase (the
pyrimidine salvage enzyme) which condenses fluorouracil with PRPP to
fluorouracil monophosphate. Fluorouracil monophosphate is
phosphorylated by nucleoside monophosphate kinase to form
fluorouracil diphosphate which is a substrate for ribonucleotide
reductase to produce 2’-deoxyfluorouracil diphosphate which is then
phosphorylated by nucleoside diphosphate kinase and hydrolyzed by
UTP phosphorylase to form 2’-deoxylfluorouracil monophosphate
which is a potent irreversible inhibitor of thymidylate synthase. This is
yet another example of a suicide inhibitor.
The mechanism for the irreversible inhibition is shown below.
O
F
HN
O
O
-O
P
O
N
S
O
OH
dUMP
ENZ
H
H
H
OH
H
:B
H
N5,N10-Methylene-THF
H2N
H
N
N
CH2
Dead End Covalent
Complex
H
H
N
H
N
O
-
O
-O
P
O
S
2
H
N
H
O
ENZ
H
OH
H
-O
P
O
CH2
F
O
O
H
O
N
S
:B
ENZ
O
OH
H
H
OH
H
H
Note that there is no proton for the general base to abstract. The formation of the covalent enzymedFUMP-THF ternary complex is irreversible.
Methotrexate and Aminopterin are potent inhibitors of dihyrofolate reductase.
CH2
HN
R
HN
H
H
N
O
OdUMP
N
N
CH
N
R
F
HN
O
H2C
O
N
H2N
CH2
III. Dietary Nucleic Acids
Nucleic acids are ubiquitous biomolecules. A significant amount of nucleic acids are ingested in our
diets. Nucleic acids are digested in our intestinal tract to nucleotides by various nucleases and
phosphodiesterases. Nucleotides are then dephosphorylated by nonspecific phosphatases.
5’-nucleotidase
NMP + H2O Nucleoside + Pi
The nucleosides are hydrolyzed by nucleosidases to release the bases.
Nucleoside + H2O base + ribose
The ribose liberated in this reaction can be catabolized for energy. And is the only portion of the
nucleotide that can be used as a source of metabolic energy.
Very little of the nucleic acids ingested in our diets become incorporated into the nucleic acids in our
cells. Most of the ingested bases are excreted.
The salvage pathways are to continuously recycle cellular constituents not incorporated dietary bases.
IV. Purine Nucleotide Catabolism
The purine nucleotides are catabolized by the following pathways. GMP is converted into guanosine by
5’-nucleotidase. Next guanosine is converted into guanine and ribose-1-phosphate by purine nucleoside
phosphorylase. Guanine is then converted into xanthine by guanine deaminase. AMP has two
degradation pathways. One pathway converts AMP into IMP by AMP deaminase. IMP is
dephosphorylated by 5’-nucleotidase to form inosine. Inosine is broken down into ribose-1-phosphate
and hypoxanthine by purine nucleoside phosphorylase. The major pathway begins with
dephosphorylating AMP by 5’-nucleotidase to form adenosine which is then deaminated by adenosine
deaminase to form inosine. Inosine is converted into ribose-1-phosphate and hypoxanthine as previously
described. Hypoxanthine is converted into xanthine by xanthine oxidase. Xanthine oxidase then oxidizes
xanthine to produce uric acid. Animals such as us excrete uric acid.
O
NH2
N
AMP
N
O
-O
N
P
O
IMP
AMP Deaminase
OH
H
OH
X
N
-O
P
O
H
H
H
H
+
H2O
OH
X
O
N
N
NH
N
O
O-
O
O
NH
Inosine
O
N
O
H
N
HO
H
H
H
H
OH
X
X
Purine Nucleoside Phosphorylase
O
N
NH
Pi
Pi
NH2
N
Adenosine
N
HO
N
Inosine
N
Adenosine Deaminase
H
H
OH
X
HO
H
+
H 2O
H
H2O2
O
H
OH
O2+
H 2O
N
O
H
NH4
N
Xanthine
Oxidase
NH
N
O
H
N
H
O
N
X
N
NH
H
N
H
N
H
Xanthine
O
O
GMP
N
O
-O
P
NH
N
O
H
OH
O
N
N
x
N
H
H
5'-NucleotidaseO
N
Pi
Guanosine
H
H
O
Pi
x
O
H
H
OH
N
O
P
O
O
N
H
+
X
H
N
H
N
H
O
Xanthine
+
H2O2
NH4
O
Guanine
H2O Deaminase
NH
N
Xanthine Oxidase
NH
N
H
Guanine
O
N
O
H
O2 + H2O
NH2
Purine Nucleoside
Phosphorylase
O
HO
Xanthine
H
OH
O
N
H
NH
N
HO
NH
NH2
H
OH
NH2
H
+
Hypoxanthine
5'-Nucleotidase
5'-Nucleotidase
H
N
NH
O
N
H
N
H
Uric Acid
O
O
H
OH
Pi
H
P
O
O
H
N
O
H
NH4
HO
O
Adenosine Deaminase
A genetic deficiency of adenosine deaminase causes severe combined immunodeficiency syndrome
(SCID). Lack of adenosine deaminase actitity causes an inability of B and T lymphocytes to proliferate
and produce antibodies. Adenosine deaminase activity is also impaired in other diseases such as AIDS,
anemia, lymphomas and leukemias. Adenosine deaminase deficiency is another target for gene therapy.
Gene therapy is the attempt to repair a genetic deficiency by the introduction of a function gene. So far
Gene therapy has experienced lots of set backs. A loss or lack of adenosine deaminase activity causes
deoxyadenosine do be converted into dAMP. dAMP is then converted in to dATP. The result is high
concentrations of dATP. dATP is a potent inhibitor of deoxynucleotide biosynthesis. Remember that
dATP binds to the activity site of ribonucleotide reductase and turns the enzyme off.
V. Pymidine Nucleoside Catabolism
Pyrimidines are also catabolized as shown.
β-Alanine is then deaminated by a transaminase to form
Malonic semialdehyde which is then oxidized and converted
into malonyl-CoA. Malonyl-CoA as a precursor for fatty
acid biosynthesis.
β-Aminoisobutryate is also deaminated by a transaminase to
form Methylmalonic semialdehyde which is then oxidized
and converted into methylmalonyl-CoA which is converted
into succinyl-CoA by methylmalonyl mutase.
VI. Regulation of Nucleotide Biosynthesis
The regulation of purine
biosynthesis is shown to the
left.
The synthesis of PRPP is
inhibited by AMP,IMP and
GMP.
PRPP is a branch point for the synthesis of pyrimidines. The first committed step for purine
biosynthesis is the formation of phosphoribosylamine. A reaction catalyzed by amidophosphoribosyl
transferase. This enzyme is allosterically inactivated by IMP, AMP and GMP. This enzyme is activated
by high concentrations of PRPP (feed forward activation). IMP is the branch point between AMP and
GMP. High concentrations of AMP inhibit adenylosuccinate synthetase. High concentrations of GMP
inhibit IMP dehydrogenase. The reaction catalyzed by adenylosuccinate synthetase activates IMP by the
transfer of the γ-phosphate of GTP. The reaction catalyzed by GMP synthetase uses ATP to activate
XMP. Thus relative high concentrations of ATP increase the rate of GMP synthesis, and relative high
concentrations of GTP increase the rate of AMP synthesis.
Regulation of Pyrimidine Synthesis
In animals pyrimidine biosynthesis is regulated by the activity of CPS II.
UDP and UTP are allosteric inactivators of CPS II. High concentrations of
ATP activate CPS II as do high concentrations of PRPP.
Orotidine-5’-monophosphate Decarboxylase in inhibited by UMP.