Nucleotide Metabolism Flashcards
Purines and Pyrimidines:
Hypoxanthine is precursor to Adenine
Xanthine is precursor to Guanine
Uracil is aminated to form Cytosine and methylated to form Thymine
Nucleoside and Nucleotide
A nucleoside is a pentose sugar that is attached in a N-glycosidic linkage to the purine or pyrimidine base
In a nucleotide a phosphate is esterified to one of the OH groupss, typically attached to the C-5’ of the nucleoside pentose.
Tide = phosphate
Synthesis of purine nucleotides via the de nova pathway
Linear synthesis pathway:
- PRPP –>* phsophoribosylamine —-> IMP#
* = first committed step in de nova synthesis
= first compound with completed purine ring
Branch point pathways:
IMP -> S-AMP -> AMP
IMP -> XMP -> GMP
Linear Synthesis
(PRPP –> IMP)
- Base is built on ribose
- 10 step energy costly process (5 ATP)
- Gutamine, glycine, aspartate provide nitrogens
- Formate and CO2 provide carbons - note that glycine also provides 2 carbons
Branch Point Synthesis
(IMP-> AMP or IMP-> GMP)
GTP provides free energy for reaction producing AMP
ATP provides free energy for reaciton producing GMP
ATP And GTP are both regulators
PRPP Synthetase
- PRPP synthetase provides substrate (PRPP) for purine de novo synthesis
* D-ribose-5-phosphate gets pyrophase (2 PO4-) - PRPP is also used elsewhere in nucleotide metabolism and in amino acid biosynthesis and so this reaction is not comitted to de novo purine synthesis
Amidophosphoribosyltransferase (ATase)
- ATase catalyzes the displacement of pyrophosphate by the amino group of glutamine.
* This reaction is the first comited (and also rate-limiting) step in the de novo synth. - ATase is subject to allosteric control by PRPP and end products of teh pathway (IMP,AMP, and GMP)
* When PRPP is high, it activates enzyme by monomerization
Synthesis and interconversion of NDPs and NTPs
NMP to NDP done by Adenylate kinase and Guanylate kinase
- AMP + ATP –> 2ADP
- GMP + ATP –> GDP + ADP
- base-specific monophosphate kinases
NDP to NTP done by nucleoside diphosphate kinase
- GDP + ATP -> GTP + ADP
- broad specificity
steady state conditions of adenylate kinase:
100X ATP: 10X ADP: 1X AMP
Purine Nucleotide Metabolism - Regulation
Controlled by relative levels of end products
- End products can regulate PRPP synthetase, ATase, and both branch point pathways (feedback regulation)
- Branch point synthesis can be stimulated by the end product of the opposite branch (cross-regulation)
Degradation and excretion of purine nucleotides
No specific order:
- Dephosphorylation removes phosphate from the pentose sugar
- Deamination removes the amino group from adenine (C-6) and guanine (C-2)
- Phosphoroylsis removes the pentose sugar
AMP -> Inosine -> Hypoxathine -> xanthine -> Uric Acid
GMP -> Guanosine -> Guanine -> xanthine -> Uric Acid
*Adenosine is potent coronary vasodilators (treat heart attack)
Xanthine dehydrogenase/oxidase (XDH/XO)
- XDH and XO convert hypoxanthine and xanthine into uric acid
- encoded by same mRNA
- XDH can be converted to XO under low O2 conditions
- By product of XO is H2O2, so not desirable
Gout - hyperuricemia
Uric acid overproduction or underexcretion
Primary gout (inherited metabolic abnormalities)
- Overexpression of PRPP synthetase
- Deficiency of hypoxanthine guanine phosphoribosyltransferase (HPRT/HGPRT)
- inhibits salvage pathway to turn Guanine -> GMP or Hypoxanthine -> AMP
- Defects in a family of renal urate transport porteins (80% of cases)
Secondary gout (acquired condition)
- Drug intake or unusual dietary habits
- meats contain a lot of nucleotides
Treatment of Gout
Use of Allopurinol that is a competitive inhibitior:
- XDH converts Allopurinol to Alloxanthine (not Xanthine)
- With less XDH available, less xanthine -> Urate production
- Can also get into salvage pathway and turn into AMP/GMP and inhibit ATase by feedback mechanism
Xanthinuria
Deficiency of Xanthine Dehydrogenase (XDH)
- reverse disorder as Gout
- You see increased levels of Guanine, Hypoxanthine and Xanthine
- No treatments - just limiting nucleotide ingestion to reduce levels
*
Immunodeficiency diseases
- A deficiency of adenosine deaminase (ADA) (removes amino group) –> T-cell and B-cell dysfunction (bubble boy)
- A deficiency of purine nucleoside phosphorylase (PNP) (removes pentose sugar) –> impairmen of T-cell function
Both of these diseases lead to 50-100 fold increased dATP or GTP levels in lymphocytes leading to inhibition of Ribonucleotide reductase (DNA synthesis, cell division)
Purine recycling (HPRT)
Hypoxanthine-guanine phosphoribosyltransferase (HPRT) can initiate Guanine and Hypoxanthine into the salvage pathway
10% of catabolized purine nucleotides are excreted as uric acid, 90% are salvaged.
The de nova pathway of purine nucleotide synthesis functions to maintain total purines by replacing that which is lost through the exrcretion of uric acid
The salvage pathway, therefore, is normally the major pathway of purine nucleotide synthesis
Deficiency of HPRT
Can lead to Lesch-Nyhan Syndrome
- HPRT is lacking or present at low levels (<1% of normal) in affected individuals
- X chromoome linked (affects males)
- High levels of uric acid produced -> gout
- Inadequate feedback inhibition of PRPP synthase and ATase leads to increased rates or PRPP synthesis and de nova purine synthesis
- GMP and AMP are not being salvaged thus are not inhibiting action of PRPP synthetase and ATase
Treatment of Lesch-Nyhan Syndrome
- Deficiency of HPRT activity is correlated with the degree of neurological complications
-
Allupurinol relieves complication of gout, no know treatment for neuro problems
- Allupurinol binds XDH to decreased Xanthine production
- Brain has 10-20 times higher HPRT activity than other tissues - lack may lead to an imbalance in purine nucleotides at critical times during development
Pyrimidine Nucleotide Metabolism - de novo synthesis
Key difference is no PRPP and uses glutamine and bicarbonate as initial substrates
A large trifunctional protein, CAD (steps 1-3), Dihydro-orotate dehydrogenase (DHODH step 4), and UMP synthase (steps 5-6)
End product being UMP which will then branch into C and T
Caramoyl Phosphate Synthetase activity of CAD
- CPS II is cytosolic and uses glutamine as the source of the amino group
- This is the rate limiting step
Conversion of UMP to UDP and UTP
- UMP is an intermediate in the synthesis of the other pyrimidine nucleotides (C and T)
- UMP + ATP –> UDP + ADP by pryimidine monophosphate kinda (specific)
- UDP + ATP –> UTP + ADP by nucleoside diphosphate kinase (broad specificity)
Synthesis of CTP
CTP synthetase converts UTP to CTP by addition of a NH2 group from Glutamine
Degradation and excretion
- Dephosphorylation removes phosphates
- Deamination removes base
- Phosphorolysis removes ribose sugar
- End products are amino acids that can be fruther metabolized
The salvage pathway
Uridine is the major recylcing molecule
- PRPP can provide ribose
Inherited Disorders
- Orotic Aciduria (UMP synthase defiecency)
- excessive orotic acid in urine and anemia
- UMP is steps 5 and 6 in de novo pathway
2. Dihydropyrimidine dehydrogenase defiency
- associated with elevated levels of uracil and thymine in body fluids and convulsive disorder
Reduction of Ribonucleotides
Occurs by Ribonucleotide Reductase (RR) at the level of diphosphates
NDP –> NTP
- An electron transfer pathway that uses NADPH to provide reducing equivalents
RR activtiy:
- allosterically regulated by dNTP.
- dATP accumulates in individuals with adenosine deaminase (ADA) deficiency
- shuts off RR for other nucleotides
Synthesis of Thymidine Nucleotides
TMP is synthesized by thymidylate synthase using dUMP as the substrate
- UDP -> dUDP -> dUMP -> dTMP -> Thymidine
- requires tetrahydrofolate as methyl transferase - controls rate of nucleotide synthesis
