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Introduction to Enzyme Catalysis

  • Two types of catalysis in enzymes:

    • Covalent Catalysis: Common in mammals and plants, used by enzymes like aldolase.

    • Metal Ion Catalysis: Utilized by fungi and bacteria.

Aldolase Mechanism

Overview of Aldolase

  • Aldolase catalyzes the cleavage of fructose 1,6-bisphosphate during glycolysis.

  • Utilizes covalent catalysis as the main mechanism.

Thermodynamics

  • To push the aldolase reaction forward:

    • Maintain a low concentration of products.

Step-by-Step Mechanism of Aldolase

  1. Binding and Conversion:

    • Substrate: Fructose 1,6 bisphosphate (F1,6BP).

    • Fructose binds to the active site and undergoes unwrapping to the linear form.

    • The binding site consists of important amino acids:

      • Lysine: Conserved across all class one aldolases.

      • Other basic amino acids and one acidic amino acid.

  2. Formation of Tetrahedral Intermediate:

    • The nitrogen atom in lysine attacks the carbonyl carbon.

    • Forms a covalent bond: Tetrahedral intermediate (with four single bonds around the carbon).

    • The carbonyl oxygen now converted to an alcohol group as the double bond is broken.

  3. Collapse of Intermediate:

    • Intermediate collapses, removing the alcohol group (via dehydration reaction):

      • Forms a carbon-nitrogen double bond (protonated Schiff base).

    • Intermediate retains a positive charge on nitrogen, critical for the next steps.

  4. Cleavage of Molecule:

    • A basic amino acid donates electrons to stabilize the alcohol group before cleavage.

    • The Schiff base acts as an electron-withdrawing group, making the bond between C3 and C4 weak, leading to its cleavage.

    • Forms glyceraldehyde 3-phosphate (GAP) as the first product.

  5. Isomerization:

    • Remaining product undergoes isomerization to form GAP (from DHAP).

    • Justification: Maintains a low concentration of GAP to drive reactions forward.

  6. Cleave Covalent Bond:

    • Water enters and performs a hydrolysis reaction, executing the reverse of the earlier steps to regenerate the enzyme and release the product.

    • Mechanism is reversible based on concentrations.

Enzyme Regulation

  • Enzymes aldolase and triose phosphate isomerase do not commonly undergo allosteric regulation.

  • Operate based on product/reactant concentrations.

Triose Phosphate Isomerization

Description

  • Converts dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GAP) using the enzyme triose phosphate isomerase.

  • Ensures both molecules can enter the payoff phase of glycolysis.

Thermodynamics and Concentration Effects

  • Delta G for GAP formation: +7.5.

  • Low GAP concentrations help move the reaction in the forward direction to proceed with glycolysis.

Summary of Key Steps in Glycolysis

Energy Investment Phase

  1. Preparation for Payoff Phase:

    • Ensure uniform concentration of GAP allowing continuation of glycolysis.

  2. Carbon Numbering:

    • Track carbons through glycolysis for clarity among products.

    • Important to maintain a consistent numbering to avoid confusion in future reactions.

Payoff Phase

Step 6: Phosphate Addition

  • Enzyme: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

  • High energy compound formation:

    • Attaching inorganic phosphate to GAP, resulting in 1,3-bisphosphoglycerate.

  • Key production of NADH (removal of hydride ion).

    • Important for energy yield and contributes to overall energetic balance in glycolysis.

Step 7: Substrate-Level Phosphorylation

  • Enzyme: Phosphoglycerate kinase.

  • Converts 1,3-bisphosphoglycerate to 3-phosphoglycerate while generating ATP.

  • Delta G: -18.8, indicates spontaneous reaction driving previous steps.

Steps 8 and 9: Phosphate Migration and Rearrangement

  • Enzyme: Phosphoglycerate mutase converts 3-phosphoglycerate to 2-phosphoglycerate:

    • Enzyme facilitates movement of phosphate, therefore making it more reactive.

  • Enzyme: Enolase further converts 2-phosphoglycerate to phosphoenolpyruvate (PEP) by removing water, forming a double bond for stability.

Step 10: Final ATP Production

  • Enzyme: Pyruvate kinase facilitates final transfer of PEP phosphate to ADP, forming pyruvate and generating ATP.

    • End step yields energetically favorable conditions aiding product formation: Delta G: -31 kJ/mol signifies high spontaneity pushing forward all previous glycolytic steps.

  • Regulation of this step is influenced by concentrations, particularly by ATP levels.

Conclusion

  • Glycolysis yields a net gain of ATP and connects to various metabolic pathways, including citric acid cycle and gluconeogenesis. Efficient regulation is critical for cell metabolism and energy balance.