Nucleotides Flashcards
What are 3 types of nucleotides in order of decreasing abundance?
1) ATP
2) Ribonucleotides (mM)
3) Deoxyribonucleotides (uM range)
What is the main structural difference between purines and pyrimidines?
Purines: bicyclic
Pyrimidines: monocyclic
What are the 3 structural components of nucleotides?
1) Inorganic phosphate
2) 5 Carbon sugar (D-ribose or 2-Deoxy-D-Ribose)
3) Nitrogenous base (purine or pyrimidine)
What is the structural difference between a nucleotide and nucleoside?
Nucleoside no phosphate, only nitrogenous base and ribose sugar
What are 2 structural components of a nucleoside and how are they bonded?
1) Nitrogenous base (purine/pyrimidine)
2) Ribose sugar (D-ribose or 2-Deoxy-D-Ribose)
- bonded by ß-N-glycosidic bond
A nucleoside is (more/less) water soluble than its free base?
More soluble
Why does coffee and tea make you feel more energetic?
Caffeine and Theophylline both inhibit Phosphodiesterase
→ prevent breakdown of cAMP (responsible for feeling energetic) to AMP
Explain the acid-base nature of nucleotides.
Strong acid
- ionisation of phosphate groups
Which nucleotide is an important methyl donor?
S-Adenosylmethionine
Which nucleotide is an important sulfur donor?
3’-Phosphoadenosine 5’-phosphosulphate (PAPS)
What are 4 examples of pyrimidines?
1) Cytosine
2) Thymine
3) Uracil
4) 5-Methylcytosine
5) Dihydrouracil (t-RNA)
6) Pseudouracil (t-RNA, eukaryotic r-RNA)
What are 4 examples of purines?
1) Adenine
2) Guanine
3) Hypoxanthine (t-RNA)
4) Xanthine (metabolism)
5) Uric acid (metabolism)
6) 7-methylguanine (5’-cap of mRNA)
Where do the C and N atoms of the purine ring come from?
C:
HCO3- → C6
Glycine → C4,5
Formate → C2,8
N:
Aspartate amine → N1
Glutamine amide → N3, N9
Glycine → N7
What are the (i) activator and (ii) inhibitor of PRPP synthesis in Eukaryotic Purine synthesis?
Activator: Pi
Inhibitor: Purine ribonucleotides (ADP, GDP)
What is needed in PRPP synthesis in Eukaryotic Purine synthesis?
Ribose-5-P → PRPP
- via PRPP synthetase
- ATP → AMP (ATP Dependent)
- Mg2+ cofactor
What are 2 ways to synthesise purine and pyrimidine ribonucleotides?
1) De novo pathway (10 steps)
2) Salvage pathways
What are the (i) activator and (ii) inhibitor of 5-phosphoribosylamine synthesis (PRPP aminotransferase rxn) in Eukaryotic Purine synthesis?
Activator: PRPP
Inhibitor: AMP, GMP
What is the moa of PABA analogs (Sulfonamide) and why do they not affect humans?
PABA analogs → CI bacterial folic acid (and thus DNA) synthesis
- CI of DHP synthase → ↓tetrahydrofolate → ↓purine synthesis
BUT humans cannot synthesise folic acid (no effect)
What is the moa of methotrexate?
Folic acid analog (anti-cancer)
- CI of dihydrofolate reductase → ↓tetrahydrofolate
→ ↓purine synthesis → ↓DNA replication
What is the moa of mycophenolic acid?
Immunosuppressant
- CI of IMP dehydrogenase
- ↓nucleic acids in T and B cells → prevent graft rejection
How are NTP, GTP and ATP (nucleoside triphosphatases) formed?
NMP → NDP → NTP
- NMP kinase → NDP kinase
- 2 ATP → 2 ADP
GMP → GDP → GTP
- GMP kinase → NDP kinase
- 2 ATP → 2 ADP
ADP→ATP
- via oxphos
Where do the C and N atoms of the pyrimidine ring come from?
C:
HCO3- → C2
Aspartate → C4,5,6
N:
Aspartate → N1
Glutamine amide → N3
NTPs are synthesised from NMPs using (specific/non-specific) NMP kinases and (specific/non-specific) NDP kinases.
NMP → specific NMP kinase → non-specific NDP kinase → NTP
How are UTP and CTP formed?
UMP → UDP → UTP
- NMP kinase → NDP kinase →
- 2 ATP → 2 ADP
UTP → CTP
- CTP synthetase
- Gln + ATP → Glu + ADP
What is the difference between CP I and II?
1) Location
I → mitochondria, II → Cytosol
2) Pathway
I → Urea cycle, → II → pyrimidine synthesis
3) N source
I → Ammonia, II → γ-amine group of glutamine
4) Regulators:
I → activated by N-acetylglutamate
II → activated by PRPP, inhibited by UTP
How does pyrimidine synthesis regulation differ in mammalian and prokaryotic (eg. E. coli) cells?
Mammalian:
- regulation at CPS II
- activator: PRPP, ATP
- inhibitor: UTP, CTP
- also regulated at OMPDC (inhibited by UMP)
Prokaryotic:
- regulation at ATCase
- activator: ATP
- inhibitor: CTP
What is orotic aciduria?
High orotate in urine
- by low activity in either (i) OMPDC or (ii) Orotate phosphoribosyltransferase (OPRTase)
- Type I: Both, Type II only OMPDC
→ megaloblastic anemia and poor growth (↓ pyrimidine synthesis)
- Rx: Uridine, Cytidine, salvage enzymes (to bypass de novo pathway)
In the biosynthesis of deoxyribonucleotides, reduction occurs at (sugar/nucleotide level)
Ribonucleotides
How does ribonucleotide reduction in deoxyribonucleotides biosynthesis in humans differ from L. leichmanii and E. coli?
Humans same as E. coli
NDP → dNDP
- ribonucleotide diphosphate reductase
- cofactor: Fe
L. leichmanii
NTP → dNTP
- ribonucleotide triphosphate reductase
- cofactor: B12, dihydrolipoate
The _______-dependant ribonucleotide reductase in eukaryote deoxyribonucleotide biosynthesis requires ________ as a reducing agent by using reducing equivalents from ________.
Fe-dependant
Requires Thioredoxin
uses NADPH
What are 3 sites for regulation of ribonucleotide reductase?
1) Activity site:
- ATP → activate
- dATP → inhibit
2) Specificity site
- determines which substrate can bind to catalytic site
3) Catalytic site
- for substrate
How are the ribonucleotides dCTP, dTTP, cGTP, dATP synthesised in exactly the same ratio (1:1:1:1)?
Start at high ATP
→ ATP occupy both activity and specificity site
→ allows reduction of CDP and UDP
→ form dCTP (1st) and dTTP (2nd) respectively
→dTTP can occupy specificity site
→ allow reduction of GDP
→ forms dGTP
→dGTP can occupy specificity site
→ allow reduction of ADP
→ forms dATP
(when dTTP, dGTP, dATP in specificity site → inhibit reduction of CDP, UDP, etc. to prevent prior rxn)
How does mammalian ribonucleotide reductase regulation differ in E. coli?
dGTP inhibits reduction of CDP and UDP in mammalian but not E. coli cells
What is the resultant molecule from all purine catabolism?
Uric acid
How is DNA catabolised into purines/pyrimidines?
1) DNA/RNA → mononucleotides
- Nucleases/ phosphodiesterases
2) Mononucleotides → nucleosides
- Mononucleotide phosphatatses
3) Nucleosides → purines/ pyrimidines
- Phosphorylases
What are 3 pathologies related to defective purine metabolism?
1) Lesch-Nyhan Syndrome (salvage)
2) Gout (degradation)
3) SCID (dNDPs)
What is Lesch-Nyhan syndrome?
Sex-linked congenital severe HGPRT deficiency (purine salvage enzyme)
- mostly males
→ accumulation of PRPP → activate de novo pathway
→ ↑ rate of purine synthesis and ↑ uric acid
→ neurological abnormality:
- mental retardation
- bizarre behaviour (aggressive, destructive, self-mutilation)
What is gout?
Painful arthritic joint inflammation due to deposition of sodium urate crystals (tophi)
- caused by ↑↑uric acid in blood and other bodily fluids
- can also cause renal calculi
- mainly males
What is the committing step of purine synthesis?
Ribose-5-P → PRPP
- via PRPP synthetase
- ATP → AMP (ATP Dependent)
- Mg2+ cofactor
What are 2 pathophysiological factors for gout and what are the implicated enzymes?
1) Impaired uric acid excretion
2) excessive uric acid production
i) HGPRT deficiency → ↑PRPP
ii) G6Pase deficiency (Von gierke’s Type I) → ↑G6P, ↑R5P, ↑PRPP, ↑uric acid, ↑lactic acid)
iii) Overactivity of PRPP synthetase
What is SCID?
Severe Combined Immunodeficiency Disease
- 30% a/w Adenosine deaminase (ADA) deficiency
→ T and B cells cannot proliferate → lack of immune response
How does adenosine deaminase deficiency lead to SCID?
↓ADA → ↑Adenosine/Deoxyadenosine
→ ↑dATP → bind to activity site of NDP reductase
→ inhibit NDP binding and reduction
→ ↓dNTP for DNA synthesis
→ ↓immune cell proliferation
How is orotic aciduria treated?
1) Uridine, Cytidine
2) salvage enzymes (to bypass de novo pathway)
What is the moa of Acyclovir?
Anti-viral
- purine analog (acycloguanosine)
→ activated by specific HSE-thymidine kinases only in infected cells
→ acyclovir phosphorylated to triphosphate form
→ Acycloguanosine substrate for HSV-specific DNApol → chain termination
What is the moa of AZT?
Anti-HIV
- AZT → AZT triphosphate → block HIV replication via RNA-dependent DNA pol
What is the moa of hydroxyurea?
Anti-cancer
- quenches free radical on NDP reductase’s catalytic site
→ ↓DNA synthesis and replication
What is the moa of 5-flurouracil?
Anti-viral
- Uracil analog
- converted to 5FdUMP → inhibit thymidylate synthase → ↓dTMP synthesis → ↓viral replication
