Transcript N kleotid Metabolizmas - mustafaaltinisik.org.uk

Nucleotide Metabolism
Pathways in nucleotide metabolism
• De novo and salvage pathways
• Nucleic acid degradation and the importance
of nucleotide salvage
• PRPP
Biosynthetic Routes:
De novo and salvage pathways
• Most organisms can synthesize purine and pyrimidrne
nucleotides from low-molecular-weight precursors in
amounts sufficient for their needs. These so-called de novo
pathways are essentially identical throughout the biological
world.
• Salvage pathways involve the utilization of preformed
purme and pyrimidine compounds that would be otherwise
lost to biodegradation. Salvage pathways represent
important sites for manipulation of biological systems.
Figure 22.1: Overview of nucleotide metabolism.
Nucleoside = Sugar + Base (no phosphate)
Nucleotide = Sugar + Base + Phosphate
Figure 4.3: Nucleosides and nucleotides
purine
pyrimidine
Nucleic Acid Degradation and the Importance
of Nucleotide Salvage
• The salvage, or reuse, of purine and pyrimidine bases involves
molecules released by nucleic acid degradation
• Degradation can occur intracellularly, as the result of cell
death, or, in animals, through digestion of nucleic acids
ingested in the diet.
• In animals, the extracellular hydrolysis of ingested nucleic
acids represents the major route by which bases and
nucleosides become available. Catalysis occurs by
endonucleases, which function to digest nucleic acids in the
small intestine. The products are mononucleotides.
• If bases or nucleosides are not reused for nucleic acid
synthesis via salvage pathways, the purine and pyrimidine
bases are further degraded to uric acid or b-ureidopropionate.
Endonuclease
Phosphodiesterase
Nucleotidase
Phosphorylase
Figure 22.2: Reutilization of purine and pyrimidine bases.
Nucleoside
phosphorylase
PRPP: A Central Metabolite in De Novo and
Salvage Pathways
• 5-Phospho-a-D-ribosyl-1-pyrophosphate (PRPP) is an
activated ribose-5-phosphate derivative used in both
salvage and de novo pathways.
PRPP
synthetase
Phosphoribosyltransferase
(HGPRT)
De novo biosynthesis of purine
nucleotides
• Purine synthesis from PRPP to inosinic acid
• Synthesis of ATP and GTP from inosinic
acid
• Utilization of adenine nucleotides in
coenzyme biosynthesis
Glycine
2 Glutamine
Asparate
10-formyl-THF
CO2
Figure 22.3: Low-molecular-weight
precursors to the purine ring.
Purine synthesis from PRPP to inosinic acid
• Purines are synthesized at the nucleotide level, starting
with PRPP conversion to phosphoribosylamine and purine
ring assembly on the amino group.
• Vertebrate cells have several multifunctional enzymes
involved in these processes.
• Control over the biosynthesis of inosinic acid is provided
through feedback regulation of early steps in purine
nucleotide synthesis. PRPP synthetase is inhibited by
various purine nucleotides, particularly AMP, ADP, and
GDP, and PRPP amidotransferase is also inhibited by AMP,
ADP, GMP, and GDP.
1. PRPP amidotransferase
2. GAR synthetase
3. GAR transformylase
4. FGAR amidotransferase
5. FGAM cyclase
6. AIR carboxylase
7. SAICAR synthetase
8. SAICAR lyase
9. AICAR transformylase
10. IMP synthase
Figure 22.4:
De novo biosynthesis
of the purine ring,
from PRPP to inosinic
acid.
Gln
Glu, PPi
PRPP amidotransferase
AMP, GMP
Gly, ATP
GAR synthetase
ADP, Pi
NH2
H2C
C
O
Glycinamide ribonucleotide (GAR)
NH2
H2C
NH2
10-FormylTHF
C
H2C
C
THF
O
CHO
O
Glycinamide ribonucleotide (GAR)
Formylglycinamide ribonucleotide (FGAR)
GAR transformylase
NH2
H2C
C
O
NH2
CHO
Gln,
ATP
Formylglycinamide ribonucleotide (FGAR)
H2C
Glu,
ADP,
Pi
CHO
C
HN
Formylglycinamidine ribonucleotide (FGAM)
FGAR amidotransferase
Trifunctional enzyme
(AICAR)
(GAR)
(FGAR)
(FAICAR)
Figure 22.5: Transformylation reactions in purine nucleotide
synthesis.
Potent inhibitors of purine nucleotide synthesis
-- structural analogs of glutamine
-- glutamine amidotransferases
Synthesis of ATP and GTP from inosinic acid
• IMP is the first fully formed purine nucleotide and is a branch
point between adenine and guanine nucleotide biosynthesis.
• The energy to drive the aspartate transfer reaction comes not
from ATP but from GTP.
• GTP accumulation would tend to promoter the pathway toward
adenine nucleotide. Also accumulation of ATP could promote
guanine nucleotide synthesis.
• The enzyme catalyzing the pathway to make AMP is inhibited
by AMP and the enzyme catalyzing the pathway to make GMP
is inhibited by GMP
IMP
dehydrogenase
GMP
AMP
Inosine
Monophosphate
(IMP)
H
XMP
aminase
Adenylosuccinate
synthetase
Adenylosuccinate
lyase
Figure 22.6: Pathways from inosinic acid to GMP and AMP.
• Nucleotides are active in metabolism primarily as the
nucleoside triphosphates. GMP and AMP are converted to
their corresponding triphosphates through two successive
phosphorylation reactions. Conversion to the diphosphates
involves specific ATP-dependent kinases.
GMP + ATP
AMP + ATP
Guanylate kinase
Adenylate kinase
GDP + ADP
2ADP
• Phosphorylation of ADP to ATP occurs through energy
metabolism, by oxidative phosphorylation, or by substratelevel phosphorylations. ATP can also be formed from ADP
through the action of adenylate kinase, acting in the
reverse of the direction.
• ATP is the phosphate donor for conversion of GDP (and
other nucleotide diphosphate) to the triphosphate level
through the action of nudeoside diphosphokinase.
GDP + ATP
GTP +ADP
• Nucleoside diphosphokinase is an equilibrium-driven
enzyme that transfers phosphate from ATP in the synthesis
of all other nucleoside triphosphates.
Purine degradation and clinical
disorders of purine metabolism
Formation uric acid
• All purine nucleotide catabolism yields uric acid.
• Purine catabolism in primates ends with uric acid, which is
excreted. Most other animals further oxidize the purine
ring, to allantoin and then to allantoic acid, which is either
excreted or further catabolized to urea or ammonia.
PNP: Purine nucleoside phosphorylase
ADA: Adenosine deaminase
(Muscle)
Nucleotidase
Nucleotidase
ADA
PNP
PNP
Guanine
deaminase
Xanthine
oxidase
Hypoxanthine
Xanthine
oxidase
Xanthine
Uric acid
Figure 22.7: Catabolism of purine nucleotides to uric acid.
Figure 22.8: Catabolism of uric acid
to ammonia and CO2
Excessive accumulation of uric acid: gout
• Uric acid and its urate salts are very insoluble. This is an
advantage to egg-laying animals, because it provides a
route for disposition of excess nitrogen in a closed
environment.
• Insolubility of urates can present difficulties in mammalian
metabolism. About 3 humans in 1000 suffer from
hyperuricemia, which is chronic elevation of blood uric
acid levels well beyond normal levels. The biochemical
reasons for this vary, but the condition goes by a single
clinical name, which is gout.
• Prolonged or acute elevation of blood urate leads to
precipitation, as crystals of sodium urate, in the synovial
fluid of joints. These precipitates cause inflammation,
resulting in painful arthritis, which can lead to severe
degeneration of the joints.
• Gout results from overproduction of purine nucleotides,
leading to excessive uric acid synthesis, or from impaired
uric acid excretion through the kidney
• Several known genetic alterations in purine metabolism
lead to purine oversynthesis, uric acid overproduction, and
gout. Gout can also result from mutations in PRPP
amidotransferase that render it less sensitive to feedback
inhibition by purine nucleotides. Another cause of gout is a
deficiency of the salvage enzyme hypoxanthine-Guanine
phosphoribosyltransferase (HGPRT).
• Many cases of gout are successfully treated by the
antimetabolite allopurinol, a structural analog of
hypoxanthine that strongly inhibits xanthine oxidase.
• This inhibition causes accumulation of hypoxanthine and
xanthine, both of which are more soluble and more readily
excreted than uric acid.
Elevated
levels
Loss of
feedback
inhibition
Decreased levels
Figure 22.9: Enzymatic abnormalities in three types of gout.
HGPRT:hypoxanthine-guanine phosphoribosyltransferase
APRT: adenine phosphoribosyltransferase
Lesch-Nyhan syndrome: HGPRT defficiency
• Lesch-Nyhan syndrome is a sex-linked trait, because the
structural gene for HGPRT is located on the X
chromosome.
• Patients with this condition display a severe gouty arthritis,
but they also have dramatic malfunction of the nervous
system, manifested as behavioral disorders, learning
disabilities, and hostile or aggressive behavior, often selfdirected.
• At present, there is no successful treatment, and afflicted
individuals rarely live beyond 20 years.
Severe combined immune deficiency (SCID)
• Patients with a hereditary condition called severe combined
immunodeficiency syndrome are susceptible, often fatally,
to infectious diseases because of an inability to mount an
immune response to antigenic chanllenge.
• In this condition, both B and T lymphocytes are affected.
Neither class of cells can proliferate as they must if
antibodies are to be synthesized. In many cases the
condition is caused from a heritable lack of the degradative
enzyme adenosine deaminase (ADA).
• The deficiency of ADA leads to
accumulation of dATP which is
known to be a potent inhibitor of
DNA replication.
• A less severe immunodeficiency results from the lack of
another purine degradative enzyme, purine nuceloside
phosphorylase (PNP). Decreased activity of this enzyme
leads to accumulation primarily of dGTP. This
accumulation also affects DNA replication, but less
severely than does excessive dATP.
• Interestingly, the phosphorylase deficiency destroys only
the T class of lymphocytes and not the B cells.
Pyrimidine nucleotide metabolism
• Pyrimidine nucleotide synthesis occurs primarily at the
free base level, with conversion to a nucleotide occurring
later in the unbranched pathway.
• Pyrimidine synthesis begins with formation of carbamoyl
phosphate. The first reaction committed solely to
pyrimidine synthesis is the formation of carbamoyl
aspartate from carbamoyl phosphate and aspartate,
catalyzed by aspartate transcarbamoylase, or ATCase.
• In enteric bacteria, this enzyme represents an example of
feedback control. The enzyme is inhibited by the end
product CTP and activated by ATP.
Aspartate
Cambamoyl
phosphate
PRPP
(glutamine)
Cambamoyl phosphate
Aspartate
PRPP
Aspartate transcarbamoylase
CTP synthetase
Figure 22.10: De novo synthesis of pyrimidine nucleotides.
Multifunctional Enzymes
in Eukaryotic Pyrimidine Synthesis
• Aspartate transcarbamoylase in eukaryotes is strikingly
different from the E. coli enzyme. In eukaryotes, the first
three reactions of pyrimidine synthesis are catalyzed by a
multifunctional enzyme, the CAD protein (Carbamoyl
phosphate synthetase, Aspartate transcarbamoylase, and
Dihydroorotase).
• In mammalian cells, reactions 5 and 6 (see Figure 22.10)
are also catalyzed by a single protein, which is called UMP
synthase.
• A site for control of pyrimidine nucleotide synthesis is the
amidotransferase, CTP synthetase, which converts UTP to
CTP.
Salvage Synthesis and Pyrimidine Catabolism
• Pyrimidine nucleotides are also synthesized by salvage
pathways involving phosphorylases and kinases,
comparable to those already discussed for purines.
• The catabolic pathways for pyrimidines are simpler than
those for purines. Because the intermediates are relatively
soluble, there are few known derangements of pyrimidine
breakdown.
• b-alanine is used in the biosynthesis of coenzyme A
Figure 22.11: Catabolic pathways in
pyrimidine nucleotide metabolism.
Deoxyribonucleotide
biosynthesis and metabolism
• Most cells contain 5 to 10 times as much RNA as DNA.
• The small fraction that is diverted to the synthesis of
deoxyribonucleoside triphosphates (dNTPs) is of
paramount importance to the cell.
• dNTPs are used almost exclusively in the biosynthesis of
DNA. There are very close regulatory relationships
between DNA synthesis and dNTP metabolism.
• DNA differs chemically from RNA in the nature of the
sugar and in the identity of one of the pyrimidine bases.
• The deoxyribonucleotide biosynthesis on two specific
processes: the conversion of ribose to deoxyribose, and the
conversion of uracil to thymine. Both processes occur at
the nucleotide level.
Ribonucleoside diphosphate
reductases (rNDP reductase):
One enzyme reduces all four
ribonucleotide to their
deoxyriboderivatives
dUMP
Thymidylate
synthase
dTMP
Figure 22.12: Overview of deoxyribonucleoside triphosphate
(dNTP) biosynthesis.
Ribonucleoside diphosphate reductase
• The enzyme catalyzing the synthesis of dNDPs from
rNDPs, reduces the hydroxyl at carbon 2’ to a hydrogen via
a free radical mechanism.
• Ribonucleotide reductase contains catalytic residues on
each of its subunits-redox-active thiols and a tyrosine free
radical stabilized by an iron-Oxygen complex.
• Hydroxyurea, an inhibitor of ribonucleotide reductase,
destroys the free radical.
rNDP reductase
a2b2, tetramer
a-subunits form R1
containing the active site.
b-subunits make up R2
containing the free radical.
A clue to the mechanism
of action of the enzyme
(tyrosine free radical)
Figure 22.13: Structure of E. coli ribonucleoside
diphosphate reductase.
Figure 22.15: Reduction of a ribonucleoside
diphosphate by rNDP reductase.
Source of Electrons for rNDP Reduction
• Electrons for the reduction of ribonucleotides come
ultimately from NADPH, but they are shuttled to rNDP
reductase by a coenzyme that is unusual because it is itself
a protein.
• Ribonucleotide reductase uses a protein cofactor,
thioredoxin or glutaredoxin, to provide electrons for
reduction of the ribonucleotide substrate.
Figure 22.16: Reductive electron transport sequences in
the action of rNDP reductase.
Table 22.1 Biological activities of thioredoxin
Regulation of Ribonucleotide Reductase Activity
• Ribonucleotide reductase has two classes of allosteric sites.
Activity sites influence catalytic efficiency, and specificity
sites determine specificity for one or more of the four
substrates.
• The activity sites bind either ATP or dATP with relatively
low affinity, whereas the specificity sites bind ATP, dATP,
dGTP, or dTTP with relatively high affinity.
• Inhibition of DNA synthesis by thymidine or
deoxyadenosine involves allosteric inhibition of
ribonucleotide reductase by dTTP or dATP, respectively
Table 22.2 Regulation of Ribonucleotide Reductase Activity
Biosynthesis of Thymine Deoxyribonucleotides
• dUMP, the substrate for thymidylate synthesis, can arise
either from UDP reduction and dephosphorylation or from
deammation of a deoxycytidine nucleotide (dCMP).
• In the reaction catalyzed by thymidylate synthase, 5,10methylenetetrahydrofolate donates both a single-carbon
group and an electron pair to reduce that group to the
methyl level.
Deoxycytidine kinase
dUTPase
dCMP deaminase
Tymidine kinase
Thymidylate synthase
Tymidine kinase
Figure 22.17: Salvage and
de novo synthetic pathways
to thymine nucleotides.
Thymidylate
synthase
Serine
transhydroxymethylase
Dihydrofolate
reductase
Figure 22.18: Relationship between thymidylate
synthase and enzymes of tetrahydrofolate metabolism.
Deoxyuridine Nucleotide Metabolism
• In addition to the biosynthetic function of dUTPase in
forming dUMP for thymine nucleotide formation, the
enzyme plays an important role in excluding uracil from
DNA.
• dUMP residues can arise in DNA not only by dUTP
incorporation but also by the spontaneous deamination of
dCMP residues.
Salvage Routes to Deoxyribonucleotide Synthesis
• Of the various deoxyribonucleoside kinases, one that
merits special mention is thymidine kinase (TK). This
enzyme is allosterically inhibited by dTTP. Activity of
thymidine kinase in a given cell is closely related to the
proliferative state of that cell. During the cell cycle,
activity of TK rises dramatically as cells enter S phase. In
general, rapidly dividing cells have high levels of this
enzyme.
• The salvage pathway to dTTP competes very efficiently
with the thymidylate synthase-mediated de novo pathway.
• Deoxycytidine kinase is also a salvage enzyme that is
feedback inhibited by dCTP. This enzyme also functions as
a deoxyadenosine kinase and a deoxyguanosine kinase.
Unlike thymidine kinase, whose activity fluctuates over the
course of the cell cycle, the activity of deoxycytidine
kinase stays relatively constant.
Thymidylate synthase: a target
enzyme for chemotherapy
• A goal of chemotherapy is to exploit a biochemical
difference between the disease process and the host tissue
in order to interfere selectively with the disease process.
• Many chemotherapeutic agents were discovered by chance,
through testing of analogs of normal metabolites. Most of
these agents are limited in their effectiveness by
unanticipated side effects, incomplete selectivity, and the
development of resistance to the agent.
• One of the most exciting areas of modern biochemical
pharmacology is drug architecture: the design of specific
inhibitors based on knowledge of the molecular structure
of the site to which the inhibitor will bind and the
mechanism of action of the target molecule.
• Inhibition of thymidylate synthase is an approach to cancer
chemotherapy, by causing specific inhibition of DNA
synthesis. Cells that are not rapidly proliferating should be
relatively immune to such agents. Thus, cancer and a wide
range of infectious diseases should be amenable to
treatment by this approach.
• Knowing the active site structure and reaction mechanism
of an enzyme allows the design of novel inhibitors, an
approach used for thymidylate synthase, but applicable to
many other drugs.
• 5-fluorouracil (FUra) and its deoxyribonucleoside, 5fluorodeoxyuridine (FdUrd) were found to be potent
inhibitors of DNA synthesis, and both are used in cancer
treatment. They are not completely selective in their effects.
• 5-fluorodeoxyuridine monophosphate (FdUMP) is a dUMP
analog that acts as an irreversible inhibitor of thymidylate
synthase.
• FdUMP is a true mechanism-based inhibitor, in that
irreversible binding occurs only in the presence of 5,10methylenetetrahydrofolate.
Figure 22.20: Mechanism for the
reaction catalyzed by thymidylate
synthase.
Figure 22.22: Orientation of substrate and coenzyme in the active
site of thymidylate synthase.
Virus-Directed Alterations of
Nucleotide Metabolism
• The T-even bacteriophages that viruses can redirect the
metabolism of their host cells.
Figure 22.23: Metabolic pathways
leading to nucleotide modifications in Teven phage-infected E. coli.
Glycosylated DNA
Other modifications made by viruses include the following:
1. Some Bacillus subtilis phages substitute uracil for thymine in
their DNA
2. Some Bacillus subtilis phages contain 5-hydroxymethyluracil in
place of thymine.
3. A phage of Xanthomonas oryzae substitutes 5-methylcytosine
for every one of the cytosines in its DNA
Biological and Medical Importance
of Other Nucleotide Analogs
• Nucleotide Analogs as Chemotherapeutic Agents
Antiviral Nucleoside Analogs
Purine Salvage as a Target
Folate Antagonists (Figure 22.18)
• Nucleotide Analogs and Mutagenesis (Figure
22.24)
• Nucleotide-Metabolizing Enzymes as Selectable
Genetic Markers
Nucleotide Analogs as Chemotherapeutic Agents
• The enzymes of nucleotide synthesis have been widely
studied as target sites for the action of antiviral or
antimicrobial drugs.
• Other analogs receiving considerable attention are those
being used to combat acquired immune deficiency
syndrome (AIDS). One such analog is AZT, the first drug
approved in the United States for the treatment of HIV
infections.
Figure 22.24: Mechanisms of mutagenesis by nucleotide analogs.
Selectable genetic markers
• Because most cells can synthesize nucleotides de novo, the
enzymes of salvage synthesis are usually nonessential for
cell viability. This means that nucleotidemetabolizing
enzymes and the genes that encode them provide selectable
genetic markers, which have a variety of uses.
• Separate salvage and de novo pathways allow selection for
survival or death of cells with particular metabolic traits.
• Modified nucleotides can be used to select cells containing
or lacking specific enzymes. Examples include the following:
6-Thioguanine selects for cells lacking an active
hypoxanthine-guanine phosphoribosyltransferase (HGPRT).
Cells containing an active enzyme convert 6-thioguanine to a
toxic compound.
• 5-Bromodeoxyuridine (BrdUrd) can be used to select cells
lacking thymidine kinase, which is needed to metabolize
BrdUrd to a toxic metabolite.
• HAT Selection - The compounds hypoxanthine,
aminopterin, and thymidine (H,A, and T, respectively) can
be used to select for cells having functional salvage
pathways.
• Aminopterin inhibits dihydrofolate reductase, which
blocks de novo purine and thymidine synthesis. Only cells
which can utilize thymidine (pyrimidine salvage) and
hypoxanthine (purine salvage) can grow in this medium.