Nucleotide Metabolism Flashcards
Which nucleotides have a double ring structure
Purines (A, G)
Nucleoside =
Base and the pentose sugar
Nucleotide de novo synthesis precursors
Amino acids
Ribose-5P
CO2
NH3
What is the same between purine and pyrimidine synthesis?
Multienzyme complex
Common precursor = phosphoribosyl pyrophosphate (PRPP)
Use ribose as the sugar
Glutamine and aspartate are the source for amino groups for the purine and pyrimidine rings
Differences between purine and pyrimidine synthesis
Glycine contributes to the purine ring only
Pyrimidine base is 1st made…and then attached to the ribose sugar (as part of PRPP)
Where…purine bases are made already associated with PRPP
PRPP synthetase
PRPP = common intermediate for both purine and pyrimidine synthesis
Phosphorylates ribose-5P with a pyrophosphate molecule derived from ATP
important regulated step in boht de novo pathways…
Activated by inorganic phosphate
Feedback inhibited by purine nucleotides
PRPP is also involved in salvage pathways
From what is ribose5P produced from?
Glucose (PPP)
Carbon sources in the purine ring
CO2 and formate
Formate donated from N-formyl H4folate
Nitrogen contribution in the purine ring?
From glutamine and aspartate
Glycine contribution to the purine ring
Carbon and nitrogen atoms
Purinosome
Multienzyme complex
10 reactions to make purines (after PRPP synthesis)
Glutamine PRPP amidotransferase
1st reaction in purinosome
Condenses the NH3 from glutamine to the C1 atom of PRPP
Product: 5’-phosphribosylamine
Liberating….PPi and glutamate
This is the beginning of the making of the purine ring attached to the ribose sugar
Commitment step and 2nd regulated step
Activated by PRPP
Feedback inhibited by endproducts of the later reacrtions, AMP, GMP, and IMP
Important cofactor in 2 steps of the purinosome overall reaction
H4Folate to supply the formyl groups
Sulfonamide drugs
Structural analogs that inhibit bacterial synthesis of folic acid…as such
They inhibit purine synthesis in bacteria
- humans do not make folate, must be obtained in diet
Methotrexate
Folic acid analog
Inhibit the regeneration (reduction) of H2Folate —> H4Folate
Inhibits purine synthesis in cancer cells, but are also toxic to all dividing cells
Final product of the purine de novo synthesis
Inosine5-monoPhosphate (IMP)
Nitrogenous base = hypoxanthine
2 additional reactions are needed to convert IMP to either AMP or GMP
AMP generation from IMP
Additions of aspartate to the inosine ring structure
Enzyme: adenylosuccinate synthetase
Fumarate is then removed in the next reaction…leaving the alpha amino group from aspartate in place
This replaces the C5 keto group with an amino group
GMP generation from IMP
Oxidation of the purine ring…making a C7 keto group
IMP dehydrogenase
An amino group donated from glutamine replaces the keto group to form GMP
AMP and GMP synthesis regulation
End products feedback inhibit the 1 st reactions in their synthesis
AMP production requires hydrolysis of GTP
While GMP needs ATP hydrolysis
So….
If [AMP] high…so is [ATP]…this will stimulate GMP production…evening the concentrations of AMP and GMP
Mycophrenolic acid
Reversible inhibitor of IMP dehydrogenase
Drug depletes purines in the T and B lymphocytes
Used as an immunosuppressant to prevent graft rejections
5’-nucleotidase
Removes the phosphate groups to generate a nucleoside in IMP, AMP, GMP degradation
1st reaction
Purine nucleoside phosphorylase
2nd reaction in IMP, AMP, GMP degradation
Removes ribose sugar (as ribose-1P) to generate the free nitrogenous base
What base does the degradation of IMP and AMP converge on?
Hypoxanthine
Both deaminated to inosine…which is then converted to hypoxanthine
Xanthine oxidase
Oxidizes hypoxanthine —> xanthine
IMP and AMP degradation
The GMP is also degradated down to xanthine…
So all 3 converge on the base xanthine
Xanthine —> uric acid
Also done by xanthine oxidase
Uric acid is readily excreted in the urine
Gout
High blood levels of uric acid (hyperuricemia)
= the end product of purine catabolism
Either the overproduction or under excretion of uric acid
Leads to deposition of urate crystals in the joints —> inflammatory response
Diagnosis requires examination of synovial fluid
Majority of the time due to lack of excretion of uric acid…can be due to processees that affect how the kidney handles urate (i.e. lactate competes for same channels that allow urate excretion)
Thiazide diuretics or exposure to lead can also affect urate excretion, leading to saturnine gout
Primary hyperuricemia
Less common cause of gout (overproduction of uric acid)
Possible X-linked PRPP synthetase mutation
Results in increase Vmax for the production of PRPP, a lower Km for ribose-5P or decreased sensitivity to purine nucleotides…its allosteric inhibitor
Probenecid or sulfinpyrazone
Increase renal excretion of uric acid
Allopurinol
Lowers uric acid snythesis
Its converted in the body to oxypurinol, a suicide inhibitor of xanthine oxidase
Reulsts in an accumulation of hypoxanthine and xanthine
—> compounds more soluble than uric acid
Adenosine daminase (ADA)
Enzyme that deaminates adenosine —> inosine
Expressed in cytosol of all cells
Humans - lymphocytes have highest activity
ADA deficiency
Accumulation of adenosine
Which is converted to its ribonucleotide or deoxyNT forms by cellular kinases
As dATP levels rise…riboNT reductase is inhibited
Which reduced ribose sugar —> 2’-deoxyribose
So… cells cannot make DNA and divide
Severe Combined Immunodeficiency Disease
Can be caused by a severe ADA deficiency
Decrease in T and B cells since cannot divide, make DNA
Purine nucleoside phosphorylase deficiency
Autosomal recessive
Like ADA deficiency also causes immunodeficiency
Less severe and only affects T cells….prompting an increase in infections
Enzymes involved in the purine salvage pathway
Adenine phosphoribosyltransferase (APRT)
Hypoxanthine-guanine phosphoribosyltransferase (HGPRT)
Both condense the free nitrogenous base and PRPP….to form AMP, IMP, or GMP
The release of the pyrophosphate and its subsequent hydrolysis make these reactions irreversible
Lesch-Nyhan syndrome
X-linked, recessive
(-) HGPRT
Results in inability to salvage hypoxanthine or guanine
Leads to excessive degradation and uric acid levels
Also causes increase PRPP and decreased IMP and GMP….therefore the glutamine PRPP amidotransferase (purine syn. Commitment step) is unregulated
Purine synthesis is increased…adds to increased degradation and increase uric acid
Uric acid stones in kidneys, urate crystals, motor dysfunction, cognitivie defects…self mutilation
Allopurinol = effective treatment … but not for mental shit
Unlike the purine ring…the pyrimidine ring
Is made before being attached to the ribose-5P
Which is then donated by PRPP
Sources of the atoms in the pyrimidine ring
Glutamine, CO2, and aspartic acid
Pyrimidine synthesis
Begins with the condensation of CO2 and an amino group from glutamine to form
Carbamoyl phosphate
Enzyme: carbamoyl phosphate synthetase II (CPSII)
….which is the isoform of the CPS1 enzyme that starts the urea cycle in mitochondria
The regulated step in pyrimidine synthesis
CPS II reaction
Feedback inhibited by UTP (end product)
Activated by the substrates ATP, PRPP
Second step of pyrimidine synthesis
Aspartate transcarbamoylase
Carbamoyl phosphate —> carbamoylaspartate
Dihydroorotase
3rd enzyme in pyrimidine synthesis
Closes the pyrimidine ring
The resulting dihydroorotate is oxidixed to produce orotic acid
Dihydroorotate dehydrogenase
Inner mitochondrial membrane
Produces ortate (orotic acid) from dihyrdroorate
Pyrimidine synthesis
**all other enzymes in this pathway are cytosolic
Orotate phosphoribosyl transferase
Produces OMP from the complete pyrimidine ring…and releases the pyrophosphate
PRPP is the ribose-5P donor
Irreversible
Orotidylate decarboxylase
OMP —> UMP
Removes the acidic carboxyl group
UMP is then phosphorylated to UDP and UTP
UMP synthase
Catalytic domains =
- Orotate phosphoribosyl transferase = makes OMP
- Orotidylate decarboxylate = makes UMP from OMP
Orotic aciduria
Deficiency in the UMP synthase
Orotic acid in the urine
Poor growth
Megaloblastic anemia
Treated with uridine which is phosphorylated to UTP…thereby inhibiting the CPSII and decreasing orotate levels
Pyrimidine synthesis regulation
CPSII = key regulated step (like the PRPP synthetase in purine synthesis)
Inihibited by UTP
Activated by PRPP
RiboNT reductase
How dNTs are made from ribo diphosphates (ADP, GDP, UDP, etc)
Reduces the 2’OH group of the riboDPs to hydrogen
Enzyme now has a disulfide bond (oxidized)
Thioredoxin reduces the enzyme back to the active form (NADPH)
Inhibited by dATP…binds to allosteric sites
—> why high dATP levels are toxic in ADA deficiency
ATP activates the enzyme
Hydroxyurea
Destroys the free radical required for the enzyme activity of riboNT reductase
Thus inhibits the generation of dNTs
Used to treat cancers, such as chronic myelogenous leukemia
Also treats sickle cell disease
Formation of deoxyThymidine
.dUDP and dCDP are phosphorylated to dCTP and dUTP…
These are converted to dUMP..which is the substrate for thymidylate synthase
To make dTMP
Adds a methyl group to the C5 atom of the uracil ring to form thymine (H4folate cofactor)
Dihydrofolate reductase and serine hydroxymethyl transferase regenerate H4folate from H2folate afterwards
Inhibited by thymine analogs such as 5-fluorouracil (5-FU) - antitumor agent
Folate deficiency
Folate is not stored in the body to any large degree
So need diet supply
Need to maintain function of cofactors such as H4Folate
Results in decrease availability of deoxyThymidine…affects DNA synthesis and repair
Associated with macrocytic anemia (large RBCs)
