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How do organic solvents affect peroxidase structure and function?

K Ryu1, J S Dordick

  • 1Department of Chemical and Biochemical Engineering, University of Iowa, Iowa City 52242.

Biochemistry
|March 10, 1992
PubMed
Summary

Organic solvents partially denature horseradish peroxidase, altering its active site and substrate binding. This significantly impacts enzyme efficiency, highlighting the role of solvent effects on substrate thermodynamics.

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Area of Science:

  • Biochemistry
  • Enzyme kinetics
  • Protein-solvent interactions

Background:

  • Horseradish peroxidase (HRP) is a crucial enzyme in various biological processes.
  • Understanding how organic solvents affect enzyme structure and function is vital for biocatalysis and pharmaceutical applications.
  • Previous studies have shown mixed effects of organic solvents on enzyme activity.

Purpose of the Study:

  • To investigate the structural and functional consequences of exposing horseradish peroxidase to various water-miscible organic solvents.
  • To elucidate the mechanisms by which organic solvents alter enzyme-substrate interactions and catalytic efficiency.
  • To determine the extent of enzyme denaturation and active site accessibility in the presence of organic solvents.

Main Methods:

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  • Enzyme activity assays were performed using horseradish peroxidase.
  • Fluorescence spectroscopy was employed to monitor changes in the tryptophan residue environment.
  • Absorbance spectroscopy and electron paramagnetic resonance (EPR) spectroscopy were used to probe active site accessibility.
  • Kinetic parameters, including Hammett rho values and Km, were determined under varying solvent conditions.

    • Main Results:

    • Organic solvents, even at low concentrations, induced partial denaturation of horseradish peroxidase, evidenced by altered tryptophan fluorescence.
    • Spectroscopic analyses revealed increased accessibility of the enzyme's active site to organic solvents.
    • Reduced local polarity in the active site enhanced hydrogen bonding between phenolic substrates and the enzyme.
    • Extreme solvent conditions significantly perturbed the reaction transition state, altering catalytic efficiencies for specific substrates.
    • Ground-state stabilization of phenolic substrates in organic media, compared to aqueous buffer, was a major factor, leading to up to a four-order-of-magnitude reduction in catalytic efficiency and increased Km values.

    Conclusions:

    • Horseradish peroxidase retains significant native active-site structure in organic media.
    • Organic solvents profoundly influence enzyme function primarily through effects on substrate thermodynamics, particularly ground-state stabilization.
    • The observed changes in catalytic efficiency and kinetic parameters underscore the importance of considering solvent effects in enzyme-catalyzed reactions involving organic media.