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Biphasic binding kinetics between FepA and its ligands

M A Payne1, J D Igo, Z Cao

  • 1Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.

The Journal of Biological Chemistry
|August 29, 1997
PubMed
Summary

Escherichia coli FepA protein binds ferric enterobactin and colicins B and D with different affinities and kinetics. Colicin B exhibits slow dissociation from FepA, enabling affinity purification.

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Area of Science:

  • Microbiology
  • Structural Biology
  • Biochemistry

Background:

  • Escherichia coli FepA is a TonB-dependent porin.
  • FepA acts as a receptor for ferric enterobactin and colicins B and D.

Purpose of the Study:

  • Characterize kinetic and thermodynamic parameters of initial ligand binding to FepA.
  • Investigate the binding stoichiometry and competition between ligands.

Main Methods:

  • In vivo binding assays to determine dissociation constants (Kd).
  • Stoichiometry and competition experiments.
  • Analysis of FepA-ligand complex dissociation rates.
  • Fluorescent FepA derivative for kinetic analysis.

Main Results:

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  • Kd values: ferric enterobactin (24 nM), colicin B (185 nM), colicin D (560 nM).
  • Ligand binding exhibits biphasic kinetics, indicating at least two binding steps.
  • Colicin B shows significantly slower dissociation from FepA compared to ferric enterobactin.
  • Ligand binding competition observed between ferric enterobactin and colicin B.

    • Conclusions:

    • FepA ligand binding involves distinct kinetic steps.
    • Differential dissociation rates of ligands from FepA.
    • Colicin B's slow dissociation facilitates FepA purification.