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Related Experiment Videos

Conformational changes affect binding and catalysis by ester-hydrolysing antibodies

A B Lindner1, Z Eshhar, D S Tawfik

  • 1Department of Immunology, The Weizmann Institute of Science, Rehovot, 76100, Israel.

Journal of Molecular Biology
|January 8, 1999
PubMed
Summary

Antibodies D2.3, D2.4, and D2.5 utilize an induced-fit mechanism for ester hydrolysis. Ligand binding triggers conformational changes, enhancing catalytic efficiency and affinity, with D2.3 showing slow isomerisation limiting its reaction rate.

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Immunology

Background:

  • Antibodies can be engineered as catalysts, mimicking enzyme activity.
  • Understanding antibody-ligand interactions is crucial for designing efficient biocatalysts.
  • Transition state analogue (TSA) immunization elicits catalytic antibodies.

Purpose of the Study:

  • To elucidate the binding kinetics and catalytic mechanisms of ester-hydrolyzing antibodies (D2.3, D2.4, D2.5).
  • To investigate the role of conformational changes in antibody catalysis.
  • To explore the influence of crystallization conditions on antibody conformation and pre-equilibrium.

Main Methods:

  • Fluorescence quenching assays to monitor antibody-TSA binding kinetics.
  • Kinetic analysis of product release to assess catalytic rates.

Related Experiment Videos

  • Analysis of crystal structures (previously determined) in conjunction with kinetic data.

    • Main Results:

    • Antibodies D2.3, D2.4, and D2.5 exhibit an induced-fit binding mechanism involving fast association and slower isomerization (k=1-7 s-1).
    • Isomerization significantly increases antibody affinity (30-170 fold) and catalytic rates.
    • Antibody D2.3 displays a slow isomerization step (k<0.02 s-1), limiting catalysis and exhibiting hysteresis.
    • Crystallization buffer, particularly polyethylene glycol, favors the active antibody conformer, influencing observed structures.

    Conclusions:

    • Antibody catalysis involves both pre-equilibrium between conformers and induced-fit isomerization.
    • The rate-limiting step in D2.3 catalysis is a slow isomerization, indicating conformational memory.
    • Crystallization conditions can artifactually stabilize the active antibody conformation, affecting structural interpretations.