Lecture 18

• Ammonia


  Incorporated into biomolecules through glutamate and glutamine, which provide the critical entry point for incorporation of NH4+ into biomolecules

  
• Glutamate


  Source of amino groups in most other amino acids

  
• Classes of glutamine synthetases


  • GSI (in bacteria)
  • GSII (eukaryotes, some bacteria)
  • GSIII (in 2 bacterial specifies)

  
• Glutamine synthetases are regulated allosterically

  
• Biological cofactors used in amino acid and nucleotide biosynthetic pathways


  • Pyridoxal phosphate (transaminations)
  • Tetrahydrofolate (1 carbon transfers)
  • S-adenosylmethionine (1 carbon transfers-methyl group)

  
• Glutamine amidotransferase


  Catalyze reactions that incorporate nitrogen derived from the amide group of glutamine

  
• 1. phosphoribosyl-1-pyrophosphate (PRPP)


  Synthesized from ribose 5-phosphate in a reaction catalyzed by ribose phosphate pyrophosphokinase, PRPP is an intermediate in several pathways

  
• The pathway to histidine in all plants and bacteria is interconnected with the purine synthetic pathway (PRPP)

  
• Allosteric regulation of amino acid biosynthesis


  The first enzyme of the pathway is allosterically inhibited by the end product (feedback inhibition)

  
• Concerted inhibition


  Regulation where products derived from a biomolecule serve as negative feedback modulators of the enzyme producing the biomolecule
• Molecules derived from amino acids


  • Neurotransmitters
  • Hormones
  • Pigments
  • Cofactors
• De novo pathways


  Lead to nucleotides and begin with metabolic precursors: amino acids, ribose 5-phosphate, CO2, NH3

  
• Salvage pathways


  Lead to nucleotides and recycle the free bases and nucleosides released from nucleic acid breakdown
• Biosynthesis of purine nucleotides


  1. Step 3: added glycine amino group is formulated by N10-formyltetrahydrofolate

  2. Step 4: glutamine contributes a nitrogen (requires ATP)

  3. Step 5: dehydration and ring closure yield 5-membered imidazole ring – AIR synthetase

  4. Step 6: a carboxyl group is added

  5. Step 7: rearrangement which transfers the carboxylate to position 4 of the ring

  6. Step 8: an amide bond forms (requires ATP)

  7. Step 9: the carbon skeleton of aspartate is eliminated as fumarate

  8. Step 10: N10-formyltetrahydrofolate contributes the final carbon

  9. Step 11: formation of inosinate (IMP)/the first intermediate with a complete purine ring

  
• Aspartate transcarbamoylase


  Catalyzes first committed step of pyrimidine nucleotide synthesis, negatively regulated by CTP

  
• Dihydroorotase


  Catalyzes removal of water from N-carbamoyl aspartate to close the pyrimidine ring

  
• Cytidylate synthetase


  Catalyzes the formation of CTP from UTP by way of an acyl phosphate intermediate (requires ATP)

  
• Pyrimidine nucleotide biosynthesis is regulated by feedback inhibition


  Aspartate transcarbamoylase (ATCase) is inhibited by end-product CTP, inhibition is relieved by ATP

  
• Regulation of ribonucleotide reductase


  Primary site determines if enzyme is active (ATP bound)/inactive (dATP bound), substrate specificity site

  
• At high dATP concentrations, a ring-shaped α4β4 structure forms

  
• Thymidylate


  Derived from dCDP and dUMP, immediate precursor is dUMP, dUTP forms by deamination of dCTP or phosphorylation of dUMP
• Thymidylate synthase


  Catalyzes the conversion of dUMP or dTMP, incorporates methyl group (atoms derived from methylenetetrahydrofolate) on pyrimidine ring

  
• Dihydrofolate reductase


  Reduces dihydrofolate to tetrahydrofolate
• Folic acid deficiency leads to reduced thymidylate synthesis, increased incorporation of dUMP into DNA, leads to DNA damage

  
• Xanthine oxidase


  Flavoenzyme that catalyzes the oxidation of xanthine to uric acid, with molecular oxygen as the electron acceptor

  
• Adenosine phosphoribosyl-transferase


  Catalyzes the reaction of a free adenine with PRPP to yield the corresponding adenine nucleotide

  
• Hypoxanthine-guanine phosphoribosyltransferase


  Catalyzes the salvage of free guanine and hypoxanthine (the deamination product of adenine)

  
• Fluorouracil


  Converted to the deoxynucleoside monophosphate FdUMP in salvage pathways, FdUMP is a suicide-based inhibitor and inhibits thymidylate synthase

  
• Glutamine Synthetase Reaction


  Glutamine synthetase catalyzes two-step reaction of NH4+ w/glutamate to produce glutamine

  1. Glutamate + ATP --> y-glutamyl phosphate + ADP
  2. y-glutamyl phosphate + NH4+ --> glutamine + Pi + H+
• Overall Glutamine Synthetase Reaction Summed


  glutamate + NH4+ + ATP --> glutamine + ADP + Pi + H+
• Biosynthesis of Amino Acids

  • Carbon skeleton precursors are derived from:
  • glycolysis
  • the citric acid cycle
  • the pentose phosphate pathway
  • Nitrogen enters by way of glutamate and glutamine
• Amino Acids - a-Ketoglutarate Metabolic Precursor

  Glutamate

  Glutamine

  Proline

  Arginine
• Amino Acids - Pyruvate Metabolic Precursor

  Alanine

  Valine

  Leucine

  Isoleucine
• Amino Acids - 3-Phosphoglycerate Metabolic Precursor 


  Serine

  Glycine

  Cysteine
• Amino Acids - Phosphoenolpyruvae and Erythrose 4-Phosphate Metabolic Precursor

  Tryptophan

  Phenylalanine

  Tyrosine
• Amino Acids - Oxaloacetate Metabolic Precursor

  Aspartate

  Asparagine

  Isoleucine

  Methioninea

  Threoninea

  Lysinea
• Amino Acid - Ribose 5-phosphate Precursor

  Histidine
• 5-phosphoribosyl-1-pyrophosphate (PRPP) synthesis & use

  1. synthesized from ribs 5-phosphate in a reaction catalyzed by ribose phosphate pyrophosphokinase
  2. PRPP intermediate in several pathways
• PRPP Synthesis Equation


  Ribose 5-phosphate + ATP --> 5-phosphoribosyl-1-pyrophosphate + AMP
• Proline Arrow Pushing Mechanism --


  Proline == cyclized derivative of glutamate