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Structure-function relationships in immobilized chymotrypsin catalysis.

D S Clark1, J E Bailey

  • 1Department of Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.

Biotechnology and Bioengineering
|April 1, 1983
PubMed
Summary
This summary is machine-generated.

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Immobilizing alpha-chymotrypsin affects its active site. Using a spacer arm during immobilization enhances enzyme activity and flexibility, confirmed by electron paramagnetic resonance (EPR) spectroscopy.

Area of Science:

  • Biochemistry
  • Enzyme immobilization
  • Spectroscopy

Background:

  • Enzyme immobilization is crucial for industrial applications, but can alter enzyme activity and structure.
  • Alpha-chymotrypsin is a widely studied serine protease.
  • CNBr-activated Sepharose 4B is a common matrix for enzyme immobilization.

Purpose of the Study:

  • To investigate the impact of immobilization conditions on alpha-chymotrypsin activity and active site configuration.
  • To compare the effects of direct coupling versus using a spacer arm for immobilization.
  • To correlate changes in enzyme activity with structural alterations using EPR spectroscopy.

Main Methods:

  • Immobilization of alpha-chymotrypsin on CNBr-activated Sepharose 4B with varying enzyme loadings and a six-carbon spacer arm.

Related Experiment Videos

  • Determination of specific enzyme activity.
  • Electron paramagnetic resonance (EPR) spectroscopy using a spin label attached to the active site.

    • Main Results:

    • Specific activity decreased with increased enzyme loading on the support.
    • Increased enzyme loading led to reduced spin label mobility, indicating restricted active site configuration.
    • Enzyme immobilized with a six-carbon spacer arm showed higher specific activity and spin label mobility compared to directly coupled enzyme.
    • EPR spectroscopy revealed structural changes in the active site consistent with activity alterations.

    Conclusions:

    • Immobilization conditions significantly affect alpha-chymotrypsin's active site structure and function.
    • The use of a spacer arm during immobilization can mitigate negative impacts on enzyme activity and flexibility.
    • EPR spectroscopy is a valuable tool for understanding structure-activity relationships in immobilized enzymes.