Lecture Notes: Enzyme Substrate Interaction and Protein Stability

Enzyme Substrate Interaction

• K on and K off:

  • K on (K1) and K off (K-) determine enzyme binding affinity (KD).
  • Lower KD indicates stronger binding. E.g., 1 nanomolar stronger than 1 micromolar.
  • Strong binding requires fast K on and slow K off.

• Protein Dynamics:

  • Proteins are not static; they can move, influencing their function and interaction.
  • Importance of considering different conformations in drug design.

Protein Structure and Stability

• Molecular Weight:

  • Average molecular weight of an amino acid is 110 Daltons.
  • Estimate protein weight: multiply number of amino acids by 110.
  • Molecular weight is expressed in kilodaltons (kDa).

• Protein Folding:

  • Nonpolar residues are interior (hydrophobic effect) and charged residues (e.g., arginine, lysine) are on the surface.
  • Polar residues can form hydrogen bonds, affecting catalytic activity.

Protein Denaturation

• Stability Factors:

  • Hydrophobic effect significantly influences protein stability through water molecule entropy.
  • Hydrogen bonds are crucial for structure but contribute little to stability.
  • Ionic interactions have minor contributions due to balancing entropy and enthalpy changes.

• Disulfide Bonds:

  • Strong covalent bonds mainly in extracellular proteins due to intracellular reducing environment.

Protein Folding Process

• Formation Process:
  • Secondary structures form first (alpha helices and beta sheets).
  • Hydrophobic collapse drives final folding; chaparones assist folding and prevent aggregation.

Protein Denaturation Agents

• Heat, pH changes, and Detergents:

  • Denature proteins by disrupting various bonds.

• Examples:

  • Browning in fruits due to polyphenol oxidase activating dopamine oxidation.

Protein Aggregation and Diseases

• Amyloidosis:

  • Insoluble protein aggregates form in tissues, leading to diseases like Alzheimer’s.

• Alzheimer’s Disease:

  • Beta amyloid plaques due to misfolded proteins.
  • Treatment strategies include inhibiting plaque formation and enhancing clearance.

Protein Therapeutics

• Insulin:

  • Early example of recombinant protein therapeutics.

• Protein Therapeutics Advantages/Disadvantages:

  • High specificity, lower toxicity, but poor oral availability.

Antibody Structure & Function

• Antibody Components:

  • Composed of light and heavy chains with variable and constant regions.
  • Variable regions determine antigen specificity.

• Limitations of Antibodies:

  • Expensive, limited to cell surface targets, not orally bioavailable.

Enhancing Therapies with Antibodies

• Antibody Drug Conjugates (ADC):

  • Deliver toxins specifically to cancer cells.

• FC Fusion Proteins:

  • Extend protein half-life by attaching to constant regions of antibodies.

Enhancing Protein Stability and Half-life

• Techniques:

  • FC fusion, PEGylation, glycosylation, and cyclization.
  • Utilize binding to serum proteins like albumin to prevent renal filtration.

• Reversible Protein Binding:

  • Use of AG10 to bind to transthyretin, enhancing half-life while maintaining activity.

Conclusion

• Next Steps:
  • Explore implications of protein misfolding in disease and potential therapeutic strategies.
  • Consider current developments and future directions in protein and antibody therapeutics.