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Related Experiment Videos

Structural bases of stability-function tradeoffs in enzymes.

Beth M Beadle1, Brian K Shoichet

  • 1Department of Molecular Pharmacology and Biological Chemistry, Northwestern University School of Medicine, 303 East Chicago Avenue S215, Chicago, IL 60611-3008, USA.

Journal of Molecular Biology
|July 30, 2002
PubMed
Summary

Enzyme active sites, crucial for function, can destabilize protein structures. Modifying key residues in AmpC beta-lactamase revealed increased stability, suggesting destabilized regions may signal binding sites.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Enzymology

Background:

  • Enzymes exhibit opposing tendencies: stable structures and functional active sites.
  • Active site formation involves exposing hydrophobic areas and specific charge/hydrogen bonding patterns.
  • Enzyme stability relies on compact structures, hydrophobic cores, and optimized hydrogen bonds.

Purpose of the Study:

  • Investigate the trade-off between enzyme stability and function.
  • Examine how substitutions in active-site residues affect AmpC beta-lactamase stability and activity.
  • Determine the structural basis for altered stability in enzyme mutants.

Main Methods:

  • Site-directed mutagenesis of key catalytic residues in AmpC beta-lactamase.
  • Enzyme activity assays to quantify changes in catalytic efficiency.

Related Experiment Videos

  • X-ray crystallography to determine the structures of mutant enzymes.
  • Thermodynamic analysis of stability changes.

    • Main Results:

    • Mutations in catalytic residues (Ser64, Lys67, Tyr150, Asn152, Lys315) reduced enzyme activity by 10^3-10^5-fold.
    • Several mutations significantly increased enzyme stability, up to 4.7 kcal/mol.
    • Crystal structures revealed stabilization mechanisms: substrate mimicry, strain relief, and improved polar complementarity.

    Conclusions:

    • Active site preorganization for function may compromise enzyme stability.
    • Destabilized regions in proteins of unknown function could indicate potential binding sites.
    • Understanding enzyme stability-function relationships provides insights into protein design and function prediction.