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Redesigning enzymes by site-directed mutagenesis.

A R Fersht, G P Winter

    Ciba Foundation Symposium
    |January 1, 1985
    PubMed
    Summary
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    Genetic engineering allows protein structure modification. Researchers engineered a more effective enzyme by studying tyrosyl-tRNA synthetase, revealing insights into enzyme catalysis and protein-DNA interactions.

    Area of Science:

    • Biochemistry
    • Molecular Biology
    • Protein Engineering

    Background:

    • Genetic engineering and recombinant DNA technology enable precise protein modification.
    • Understanding enzyme-substrate interactions is crucial for catalysis and specificity.
    • Tyrosyl-tRNA synthetase from Bacillus stearothermophilus serves as a model system.

    Purpose of the Study:

    • To systematically analyze protein structure-function relationships using site-directed mutagenesis.
    • To investigate the role of hydrogen bonding in enzyme catalysis and specificity.
    • To engineer enzymes with improved substrate affinity and novel properties.

    Main Methods:

    • Site-directed mutagenesis of tyrosyl-tRNA synthetase genes.
    • Kinetic analysis of mutant enzymes.

    Related Experiment Videos

  • Protein X-ray crystallography for structural data.
  • Analysis of energetics via solvent water exchange reactions.

    • Main Results:

    • Fine-structure analysis revealed subtle roles of hydrogen bonding in catalysis.
    • Modification of ATP- and tyrosine-binding residues elucidated energetic contributions.
    • An engineered enzyme with significantly enhanced enzyme-substrate affinity was successfully created.
    • Catalysis of Tyr-AMP formation does not rely on classical acid-base or covalent mechanisms.

    Conclusions:

    • Site-directed mutagenesis combined with structural data provides direct insights into enzyme mechanisms.
    • Protein engineering holds promise for creating enzymes with tailored specificities, activities, and structures.
    • Enzyme catalysis may involve stabilization of reaction intermediates rather than classical mechanisms.