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Related Experiment Videos

Arthrobacter D-xylose isomerase: protein-engineered subunit interfaces

L Varsani1, T Cui, M Rangarajan

  • 1Centre for Biotechnology, Imperial College of Science, Technology and Medicine, London, U.K.

The Biochemical Journal
|April 15, 1993
PubMed
Summary
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Mutating Arthrobacter D-xylose isomerase at subunit interfaces did not affect enzyme activity or stability. However, a specific Tyr253 mutation reduced thermostability and altered metal ion binding, impacting enzyme function.

Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Engineering

Background:

  • D-xylose isomerase is a crucial enzyme in carbohydrate metabolism.
  • Understanding enzyme stability and activity is vital for industrial applications.
  • Protein engineering allows modification of enzyme properties.

Purpose of the Study:

  • To investigate the impact of engineered disulfide and salt bridges on Arthrobacter D-xylose isomerase.
  • To analyze the effects of specific mutations on enzyme stability and catalytic activity.
  • To elucidate the structural and mechanistic basis of D-xylose isomerase inactivation.

Main Methods:

  • Site-directed mutagenesis to introduce disulfide and salt bridges.
  • Enzyme activity assays across various pH and metal ion conditions.

Related Experiment Videos

  • Thermostability and urea denaturation studies.
  • X-ray crystallography to determine enzyme structure.

    • Main Results:

    • Mutations at A-A* interfaces showed no change in activity or stability.
    • A Tyr253 mutation (Y253C) at the A-B* interface reduced thermostability and urea stability.
    • X-ray crystallography revealed conformational changes in Glu185 and Asp254 in the Y253C mutant.
    • The Y253C mutant exhibited altered kinetic parameters (Km, Vmax) and pH-dependent activity changes with different metal ions.
    • Electrostatic repulsion and altered metal ion binding at Site [2] were implicated in the Y253C mutant's reduced activity.

    Conclusions:

    • Engineered inter-subunit bridges at A-A* interfaces do not significantly alter D-xylose isomerase properties.
    • The Tyr253 mutation introduces conformational instability, affecting enzyme thermostability and metal ion coordination.
    • Subunit dissociation is not the primary mechanism of thermal inactivation; active site conformational changes initiate unfolding.
    • The study provides insights into the structural determinants of D-xylose isomerase stability and catalytic mechanism.