notes lecture 18

Overview of Aldolase and Related Reactions

General Information on Aldolase and Reactions Involved

  • The transcript discusses the aldolase enzyme, specifically human and bacterial aldolase, detailing their mechanisms and implications in metabolic pathways.

  • Key compounds focused on include dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP).

  • The aldolase reaction is essential in converting these intermediates in glycolysis.

Mechanism of Aldolase Reaction

  • Description of the aldolase reaction involving formation of a covalent Schiff base:

    • The mechanism follows a different pathway compared to other aldolases, leading to the production of a more stable enamine intermediate rather than an enolate intermediate.

Formation of Nucleophilic Carbanion

  • If designing an enzyme:

    1. Include an active site side chain that acts as a general base, abstracting a proton.

    2. Have an oxyanion hole to stabilize the enolate resonance form.

  • Importance in Bacterial Aldolase:

    • A bound Zn2+ polarizes the C=O, stabilizing the enolate form.

  • Unique Strategy in Human Aldolase:

    • Human aldolase employs covalent catalysis, which results in a lower transition state energy and a pathway involving a more stable enamine intermediate.

Formation of Schiff Base (Iminium Ion)

  • The Schiff base formation occurs as follows:

    • Begins with the covalently attached enamine intermediate to the enzyme (Lys residue)

    • The intermediate is represented as:
      C=NH−Lys−EnzC = NH - Lys - Enz with further necessary transformations leading to the reduction of energy and stabilization of the reaction.

  • The importance of protonated Schiff base (iminium ion) in catalysis is emphasized.

Comparison of Aldolase Structures

  • Stability Comparison:

    • Human aldolase's enamine intermediate is more stable than the corresponding enolate intermediate derived from base-catalyzed cleavage.

  • Summary of reaction pathway:

    • Formation occurs via enamine as opposed to direct aldol cleavage observed in bacterial aldolase.

    • The transformation steps leading to the final products illustrate the intricate connections within biochemical pathways.

Glycolytic Pathway Progression After Aldolase Reaction

  • Aldolase reaction produces GAP and DHAP:

    • GAP progresses towards pyruvate directly.

    • DHAP (ketose) is converted to GAP, facilitated by Triose Phosphate Isomerase (TPI).

  • Function of TPI:

    • Catalyzes interconversion of ketose and aldose, similar to phosphohexose isomerase.

Reaction Models and Themes

  • Recap of previous reactions discussed:

    • 5 key reactions identified in glycolysis:

    • 2 reactions catalyzed by kinases

    • 2 reactions involving aldose-ketose isomerization

    • 1 aldolase-mediated reaction

Glycolysis Energy Considerations

  • The investment phase in glycolysis sees:

    • Input: Glucose + 2 ATP --> 2 GAP + 2 ADP

    • Being discernible that there is no ATP produced at this stage; two ATP units are used.

  • Subsequent Steps to Produce Pyruvate:

    • Following GAP formation, five more steps are necessary to convert GAP to pyruvate, highlighting the complexity of glycolytic transformations.

Key Subsequent Reactions: Conversion and Production

  • First Step Post-Aldolase Reaction:

    • Converting GAP into a high-energy phosphodonor using oxidation:

    • The reaction involves Pi and NAD+ transforming into NADH and H+.

    • Key Product: 1,3-bisphosphoglycerate (1,3-BPG).

  • Further elaboration expected in subsequent lectures regarding this reaction and its implications in glycolysis.

Final Steps in Glycolysis to Pyruvate Production

  • The remaining enzymatic reactions include:

    1. Reaction with Phosphoglycerate Kinase:

      • Conversion of 1,3-BPG into 3-phosphoglycerate (3-PG), yielding ATP.

    2. Phosphoglycerate Mutase Reaction:

      • Transformation of 3-PG into 2-phosphoglycerate (2-PG).

    3. Enolase Action:

      • Conversion of 2-PG into phosphoenolpyruvate (PEP), with water lost.

    4. Pyruvate Kinase Reaction:

      • Final production of pyruvate from PEP, resulting in a substantial net gain of ATP from initial glucose and ATP investment.

Summary of Inputs and Outputs in the Pathway

  • Conclusively noted:

    • Final equation for the glycolytic pathway:

    • 2 ATP and 2 NADH produced from the conversion of one glucose to 2 pyruvate, with an investment of 2 ATP initially.

    • The transformation of biochemical intermediates emphasizes the intricate nature of metabolic pathways and energy yield with glycolysis at its forefront.