Nucleotide Biosynthesis

Nucleotide Biosynthesis Notes

Review of Previous Topics

  • Previous lectures covered:
    • Metabolism of carbohydrates.
    • Lipid metabolism:
      • Lipids serve as energy storage molecules (storage fats).
      • Used for membrane lipid biosynthesis.
      • Fatty acid breakdown and resynthesis.
    • Amino acids as an energy source:
      • Breakdown pathways for amino acids.

Introduction to Nucleotide Biosynthesis

  • Instead of amino acid biosynthesis, the lecture will focus on nucleotide biosynthesis.
  • Nucleotides:
    • Important class of biomolecules.
    • Involved in information storage (DNA and RNA).
    • Every cell requires a copy of its genome encoded on DNA.
    • DNA duplication is crucial for cell division, especially in rapidly replicating cells, necessitating a balance of nucleotides.

Nucleotide Structure

  • Nucleotides are composed of:
    • A sugar (pentose):
      • Pentose sugars are essential for DNA and RNA biosynthesis.
      • DNA contains deoxyribose, while RNA contains ribose.
      • DNA (two-deoxyribose) is more stable than RNA because the hydroxyl group in RNA can cleave the polymer.
    • Phosphates:
      • A nucleoside becomes a nucleotide upon the addition of a phosphate.
      • Three common types: nucleotide monophosphate (NMP), diphosphate (NDP), and triphosphate (NTP).
      • NTPs contain a nitrogenous base, which dictates how they interact with each other.
    • A nitrogenous base:
      • Purines and pyrimidines are the two types of nucleobases.
      • Both contain nitrogen atoms connected by single carbon bridges.
      • Purines attach to the sugar at one position, while pyrimidines attach through a nitrogen atom.

Nucleobases

  • Nitrogenous bases found in biomolecules:
    • More than adenine and guanine exist.
    • Adenine and guanine are derived from inosine.
    • Uracil, cytosine, and thymine.
    • Oratate serves as a precursor to these bases.
  • Hydrogen bonding:
    • Dictates DNA assembly and replication.
    • Hydrogen bond donors have a hydrogen atom attached.
    • Hydrogen bond acceptors have lone pairs of electrons.
    • Adenine pairs with thymine, and guanine pairs with cytosine.

Nucleotide Metabolism

  • Essential for both catabolic and anabolic pathways.
  • Catabolic pathways are less emphasized because nucleobases are primarily recycled rather than used for energy.

Catabolic Pathways

  • Nucleotide catabolism results in:
    • Breaking down DNA or RNA polymers into individual monomers.
    • Cleavage of the phosphate group by nucleotideases.
    • Nucleosidases cleave off the base, leaving ribose.
    • Ribose enters the non-oxidative pentose phosphate pathway.
  • Nucleobases are mostly recycled.
  • Adenine is converted to inosine before breakdown, unlike guanine.

Nucleotide Biosynthesis Pathways

  • Two pathways for nucleotide biosynthesis:
    • Salvage pathway:
      • Recycles bases from the diet.
      • Involves activating a ribose sugar.
      • Starts from ribose-5-phosphate, which comes from the oxidative pentose phosphate pathway.
      • Ribose-5-phosphate is activated using ATP to form PRPP (5-phosphoribosyl-1-pyrophosphate).
      • Base attacks PRPP, kicking off pyrophosphate to form a nucleotide.
      • Energy-intensive, using two ATP equivalents to activate.
      • PRPP also used in de novo pathway.
    • De novo pathway:
      • Synthesizes bases from scratch.
      • Builds the purine base directly on the sugar, starting with PRPP.

De Novo Purine Biosynthesis

  • Builds the base directly on the sugar.
  • First step: transfer of nitrogen onto PRPP.
  • Amino acids in purine biosynthesis:
    • Glutamine donates an amino group.
    • Glycine and aspartate are directly used.
    • CO2_{2} incorporation from bicarbonate.
    • Formyl group from formyl tetrahydrofolate.

Role of Glutamine

  • Glutamine acts as an activated ammonia donor.
  • Glutamine vs. Glutamate:
    • Glutamine is similar to glutamate but with an extra nitrogen.
    • Glutamine is made from glutamate to activate ammonia.

De Novo Purine Biosynthesis Steps

  • Nitrogen atoms come from glutamine.
  • Formate groups come from formyl THF, which comes from methylene tetrahydrofolate, derived from serine or glycine.
  • Five metabolites are required and are derived or can be derived from amino acids.
  • PRPP is an intermediate in both salvage and de novo biosynthesis.

Glutamine Amidotransferases

  • Transfer NH3NH_{3} groups.
  • Break the amide bond of glutamine using a thiol in the active site, releasing NH3NH_{3}
  • This occurs in a hydrophobic channel, excluding water.
  • The enzyme consists of two active sites connected by a hydrophobic tunnel.

First Step in Purine Biosynthesis

  • Starting with PRPP, the product is 5-phosphoribosylamine.
  • The enzyme is glutamine PRPP amidotransferase.
  • SN2 displacement reaction kicking off the pyrophosphate.

Glycine Activation

  • Glycine is activated by ATP to create a better leaving group.
  • Glycine-ATP mixed anhydride intermediate.
  • Amine attacks, releasing phosphate and forming an amide bond.
  • The enzyme responsible is GAR synthetase, producing glycinamide ribonucleotide (GAR).

Transformylase Reaction

  • Using formate.
  • GAR transformylase: GAR + formate.
  • Formate comes from methylene tetrahydrofolate formed from glycine or serine.
  • Formal tetrahydrofolate donates the formate group through a nitrogen group exchange reaction.

Cyclization and Amide Activation

  • Cyclization through activation of amide.
  • Amides are unreactive due to resonance, where electron density is shared.
  • Amid bonds are planar.
  • Oxygen is made a better leaving group by transferring a phosphate from ATP.
  • FGAR amidotransferase converts FGAR to FGAM.
  • NH3NH_{3} from glutamine attacks and replaces the phosphate.

FGAM Cyclase

  • FGAM cyclase facilitates cyclization.
  • Textbook diagram is incorrect; oxygen is lost as phosphate, not water.
  • Phosphate transfers on molecule.

Carboxylation and Nitrogen Transfer

  • Air is carboxylated using CO2CO_{2}.
  • The carboxylated form of the air molecule is called CARE.
  • ATP is used to activate the carboxylate group, and a nitrogen group from aspartate is transferred.
  • Elimination reaction removes the rest of aspartate, leaving fumarate, which enters the citric acid cycle.

Ring Closure and IMP Formation

  • Formyl tetrahydrofolate is used to add the last carbon.
  • Amine group exchange reaction.
  • Final cyclization occurs spontaneously with the loss of water.
  • The result is inosine monophosphate (IMP), which must be converted to the actual nucleotides.

Conversion of IMP to AMP and GMP

  • The enzyme phosphorylates.
  • The phosphorylation gets a direct displacement reaction replacing it with a Nitrogen from aspartate.
  • To make AMP from IMP, GTP is used.
  • To make GMP, ATP is used.
  • They regulate each other.

Key Takeaways of De Novo Purine Biosynthesis

  • De novo purine nucleotide synthesis uses PRPP and multiple enzymes to construct the base.
  • It utilizes glycine, glutamine, aspartate, bicarbonate, and formate (from N10-formyl THF).
  • The starting with the addition of PRPP needs two ATPs and needs 5 ATPs for the process
  • The IMP needs one more ATP to be converted to either AMP or GMP.
  • Multi enzyme complexes are generated irreversibly.

Purinosomes and Metabolic Clusters

  • Intermediates in purine nucleotide biosynthesis have highly reactive groups, requiring sequestration.
  • Enzymes colocalize to form purinosomes to minimize off-target reactions.
  • Complexes are liquid liquid phase separated and depend on cellular needs.
  • This was the first example of metabolomes or metabolite prosthetic pathways.

Historical Context: Benkovich Lab and Song An

  • Steven Benkovich's lab investigated these reactions and enzyme complexes.
  • Song An from UMBC (originally a postdoc in Benkovich's lab) discovered that these enzymes colocalize and form reversible subcellular organelles.