Lecture 5: Glycolytic Reactions - Stage 2

Final Reactions of the Glycolytic Pathway

Overview of Glycolytic Pathway

  • The glycolytic pathway consists of two main stages:
    • Stage One: Investment Phase
    • Two moles of ATP are invested per mole of glucose.
    • This investment results in the production of two moles of glyceraldehyde 3-phosphate (GAP).
    • Stage Two: Payoff Phase
    • A total of four moles of ATP are produced, resulting in a net gain of two moles of ATP after accounting for the two moles invested in Stage One.
    • Other products include two NADH and two pyruvate molecules, which are essential for subsequent cycles (e.g., citrate cycle).

Stage Two - Payoff Phase Details

  • Net ATP Gain
    • Total ATP produced: 4 moles
    • Net ATP after investment: 2 moles
  • Key Outputs of Stage Two:
    • 2 NADH (which can later contribute to ATP production in aerobic respiration)
    • 2 Pyruvate (important for the citrate cycle)

Reaction Six: Glyceraldehyde 3-Phosphate Dehydrogenase

  • Process
    • Reactants: Glyceraldehyde 3-Phosphate (GAP) + Inorganic Phosphate (Pi)
    • Product: 1,3-Bisphosphoglycerate (1,3-BPG) + NADH
  • Mechanism
    • GAP undergoes oxidation with the help of NAD+, which is reduced to NADH.
    • Inorganic phosphate (Pi) contributes to the phosphorylation of GAP.
  • Notes on Phosphate Source
    • Inorganic phosphate comes from the environment (abundant in water) rather than from ATP.
    • The phosphorylating product is highly reactive, facilitating subsequent steps in glycolysis.

GAPDH Mechanism Explained

  • Enzyme Active Site Interactions
    • GAP enters the active site along with NAD+.
    • A nucleophilic attack occurs by a cysteine residue from the enzyme, leading to a covalently bound intermediate (hemithioacetal).
    • NAD+ accepts a hydride ion from the substrate, forming NADH and facilitating product formation.
  • NADH Production
    • NADH is a high energy molecule and will exit the active site post-reaction, allowing another NAD+ and inorganic phosphate to enter.
    • The phosphates engage in nucleophilic attacks to generate the 1,3-BPG product.

Metabolite Energetics in Glycolysis

  • High-energy intermediates drive ATP synthesis via substrate-level phosphorylation following the free energy changes during reactions.
  • Key Intermediates
    • Phosphoenolpyruvate (PEP): High energy substrate for reaction 10, $ ext{ΔG}^ ext{o} = -61.9 ext{kJ/mol}$.
    • 1,3-Bisphosphoglycerate: From reaction 7, $ ext{ΔG}^ ext{o} = -49.4 ext{kJ/mol}$.
  • ATP Energy Levels
    • Generation of ATP from these high-energy intermediates occurs through the phosphorylation of ADP, leveraging the standard Gibbs free energy changes.

Reaction Seven: Substrate Level Phosphorylation

  • Conversion
    • From: 1,3-BPG
    • To: 3-Phosphoglycerate (3PG) using the enzyme Phosphoglycerate Kinase
  • Significance of Substrate-Level Phosphorylation
    • ATP is produced without the need for ATP synthase, distinguishing it from oxidative phosphorylation.

Reaction Eight: Phosphoglycerate Mutase

  • Mechanism
    • 3-Phosphoglycerate is transformed into 2-Phosphoglycerate through a phosphate group transfer.
    • The enzyme contains a phosphohistidine that facilitates this phosphoryl transfer reaction.

Reaction Nine: Enolase

  • Water Removal
    • Conversion of 2-Phosphoglycerate to Phosphoenolpyruvate (PEP) occurs via dehydration.
    • PEP becomes a strong phosphate donor due to the high potential energy associated with its structure.

Reaction Ten: Final Steps and Net ATP Production

  • ATP Generation
    • PEP donates its phosphate to ADP, resulting in a second instance of substrate-level phosphorylation, yielding a total of 4 ATP (2 net ATP after accounting for investment).
  • Summary of ATP Production
    • Total ATP Generated: 4 moles per glucose molecule
    • Net ATP after investments: 2 moles per glucose molecule.

Conclusion

  • The glycolytic pathway, through various reactions, leads to both energy (ATP, NADH) generation and necessary substrates for further metabolic cycles, enhancing the overall energy yield from glucose.

Hasta luego, Wildcats.