Nucleotide Metabolism I & II

Question 1

Severe Combined Immunodeficiency Disorder

SCID

Answer 1

1. Deficiency in adenosine deaminase involved in AMP degradaion pathway
2. Individual has no functional immune system: Lymphocytes do’t develop properly
3. Mainly in T cells b/c they contain an active kinase that converts dADP → dATP
4. In T lymphocytes, leads to 100x increase in dATP levels → inhibits ribonucleotide reductase → other dNTPs are decreased in T cell

• high AMP → ADP → dATP
• dATP inhibits reductase at primary site levels dCTP, dTTP, dGTP are deficient
• prevents cell replication and lymphocyte proliferation

Question 2

Lesch-Nyhan Syndrome

Answer 2

1. Deficiency in nucleotide salvaging enzyme hypoxanthine-guanine phosphoribosyltransferase
2. Neurological: Mental retardation, spasticity, irregular and jerky movements, self-mutilation (chew fingers)
   * In having to rely on de novo synthesis, brain is unable to maintain levels of ATP required for signaling
3. Hyperuricemia: too much uric acid in blood → gout caused by accumulaiton of PRPP leading to purines
   * Hypoxanthine gets shunted to catabolism
4. Almost exclusively males; x chromosome

Question 3

Plasticity

Answer 3

Ability to be changed into different molecules

1. RNA’s 2’OH group allows it to act as a nucleophile to cleave phosphodiester bonds and splicing → unstable
2. DNA is more stable and effectively promotes replication

ATP is a ribonucleotide

dATP = deoxyribonucleotide (lacks 2’OH)

Question 4

Major nucleotide fxns

(6)

Answer 4

1. precursors/substrates for DNA and RNA synthesis
2. Carriers of chemical energy (ATP major energy carrier)
3. Cofactors: NAD, FAD, SAM
4. activated biosynthetic intermediates (UDP-GLC)
5. Signal molecules (2nd messengers cAMP)
6. Covalent modification of enzymes (ADP-ribosylation) cholera toxin in SI epithelial cells prevents bound GTP from become GDP, Gs constantly activates adynylyl cyclase → increase cAMP → secrete Cl → severe diarrhea →dehydration & death

ATP >> GTP > CTP/UTP

Question 5

Nucleotide synthesis: de novo

PURINES

(5)

Answer 5

1. ribose-5-phosphate + ATP → PRPP + AMP

ribose phosphate pyrophosphokinase (PRPP synthetase)

2. PRPP + gln→ 5-phosphoribosylamine +glu + PPi

glutamine-PRPP amidotransferase *committed 1st step*

3. 5-phosphoribosylamine → GAR→FGAR→ AIR →CAIR AICAR→IMP

• Glycine adds 3 atoms *ATP
• N-formylTHF (folate) single C transfer
• HCO₃⁺ or CO₂ *ATP
• Aspartate adds Nitrogen*ATP (→fumarate)
• N-formylTHF

4. IMP → AMP or GMP

• AMP: Asp + GTP (adenylosuccinate synthetase & lyase) → fumarate
• GMP: H2O + NAD / Gln + ATP (IMP DH & XMP-gln amidotransferase)

5. AMP/GMP → ATP/GTP *Kinases

Question 6

Nucleotide synthesis: de novo

PYRIMIDINES

Answer 6

1. HCO₃^- + Gln + ATP → Carbamoyl phosphate

Carbamoyl phosphate synthetase II *Committed step

2. Carbamoyl phosphate → carbamoyl aspartate
3. Carbamoyl aspartate → dihidroorotate → orotate
4. Orotate + PRPP → orotidylate → UMP → UTP → CTP

• Orotate phosphoribosyl transferase
• orotidylate decarboxylase (→CO2)
• kinases
• CTP synthetase (gln + ATP)

Question 7

Nucleotide metabolism: feedback inhibition

Answer 7

1. ribose-5-phosphate → PRPP
   * Inhibited by: ADP, AMP, GMP, IMP
2. PRPP → 5-phosphoribosylamine

• Activated by PRPP (allosteric)
• Inhibited by: AMP, GMP, IMP

3. IMP → XMP
   * Inhibited by GMP
4. IMP → Adenylosuccinate
   * Inhibited by AMP

1. Carbamoyl phosphate → carbamoyl aspartate

• Activated by ATP + PRPP
• Inhibited by CTP (pro), UDP + UTP (euk)

2. Orotidylate (OMP) → UMP
   * Inhibited by UMP

Question 8

Myoadenylate deaminase deficiency

Answer 8

1. Deficiency in AMP deaminase
2. Can’t exercise for a long time because patient can sustain energy needs

AMP → IMP allows adenylate kinase to move rxn to the right

2 ADP ⇔ ATP + AMP

*supplies a burst of ATP during muscle exercise

Question 9

Ribonucleotides → deoxyribonucleotides

Answer 9

ribonucleotide reductase: NDP → dNDP

*Reducing power ultimately comes from NADPH

Regulation:

1. Primary regulaiton site: ATP (on), dATP (off)
2. Substrate specificity site: In order to get balanced levels of dNTPs from NTPs, priority is given to NTP at lowest concentration

Question 10

Thymidyate formation (dTMP)

Answer 10

UDP → dUDP → dUTP via kinase dCTP → dUTP via deaminase

1. dUTP → dUMP
   * dUTPase (cleaves PPi)
2. dUMP + N5,N10-methylene-THF→ dTMP

• thymidylate synthase
• THF is regenerated by DHF reductase *NADPH & serine hydroxymethyltransferase *PLP

Question 11

Purine catabolism

Answer 11

When there is a build up of purine nucleotides, need to degrade and prepare for excretion

1. Dephosphorylation w/ **5’-nucleotidase **

*adenosine → inosine via **adenosine deaminase **

2. Sever base & ribose w/ nucleosidase
3. *Guanine can be released into blood or converted to xanthine via **guanine deaminase ***Hypoxanthine can be released into blood or converted to xanthine via oxidase
4. xanthine → uric acid w/ xanthine oxidase(H₂0→H₂O₂) H₂O₂ eliminated by scavenging enzymes

Question 12

Pyrimidine catabolism

Answer 12

1. Nitrogens are relased as ammondium ions or transferred to α-keto acids to produce AA
2. Disposed ultimately in the form of urea

Question 13

Nucleotide synthesis: salvaging pathway

PURINES

Answer 13

1. Diet → adenine + PRPP → AMP + PPi *requires 1ATP

***Adenosine phosphoribosyltransferase **

2. Hypoxanthine or Guanine + PRPP → IMP/GMP + PPi

*Hyoxanthine-guanine phosphoribosyltransferase

HGRTase

Neural cells are dependent on this pathway

Question 14

Gout

Answer 14

1. High levels of uric acid in blood due to excretion problem or too much purine catabolism in liver
2. In blood pH, uric acid is negative and forms salt crystals (sodium urate)
3. WBC take sodium urate up, and secrete hydrolytic enzymes that can damage collagen → inflammation and pain in joints (can also be basis of forming kidney stones)
4. Treatment: allopurinol inhibits xanthine oxidase (competitive inhibitor) & avoiding foods rich in nucleotides

*However, guanine can still be converted to xanthine

Question 15

Cancer & Viral infections

(4)

Answer 15

1. Anti-cancer therapy: inhibit DNA synthesis

• Glutamine analogs inhibit gln amidotransferases: azaserine & acivicin
  
  • blocks nitrogen donor steps in nucleotide biosynthesis
  • Problem: very toxic agents & also affects nrg metabolism, managing Nitrogen levels
• Hydroxyurea inhibits ribonuclotide reductase
  
  • targets dNTP synthesis only
• Flourouracil → FdUMP serves as a suicide substrate to thymidylate synthase
  
  • uracil analog
  • prevents hydride shift
• Folate analogs methotrexate and aminopterin inhibit thymidylate synthase
  
  • Competitive inhibitors of DHF reductase
  • high levels are required to constantly block

2. Anti-viral therapy: Inhibit replicatin of viral genome while minimizing detrimental effects on uninfected cells