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Related Experiment Videos

Engineering a novel specificity in subtilisin BPN'

M Rheinnecker1, G Baker, J Eder

  • 1MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, University Chemical Laboratory, U.K.

Biochemistry
|February 9, 1993
PubMed
Summary
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Single amino acid changes in subtilisin BPN

Area of Science:

  • Enzymology
  • Protein Engineering
  • Biochemistry

Background:

  • Subtilisin BPN' is a widely studied serine protease.
  • Enzyme specificity is crucial for catalytic efficiency and substrate recognition.
  • Modifying enzyme active sites can alter substrate preference.

Purpose of the Study:

  • To improve the specificity of subtilisin BPN' for substrates with large hydrophobic P4 residues.
  • To investigate the structural and catalytic effects of mutations at positions 104 and 107.
  • To engineer enhanced substrate selectivity in proteases.

Main Methods:

  • Site-directed mutagenesis was used to create mutant subtilisins.
  • Mutations targeted positions 104 (Tyr to Phe) and 107 (Ile to Val, Ala, Gly).

Related Experiment Videos

  • Enzyme kinetics (kcat/KM) were measured to assess substrate specificity.

    • Main Results:

    • Mutant subtilisins, particularly I107G, showed significantly increased specificity for large hydrophobic P4 residues (>200-fold improvement).
    • The I107G mutation caused a catalytic defect for small substrates, reducing activity by 93%.
    • This catalytic defect was compensated by longer substrates with large P4 residues, restoring specificity.

    Conclusions:

    • Single amino acid substitutions can dramatically enhance protease specificity.
    • Mutations at position 107 can create a catalytic lesion that is substrate-dependent.
    • Engineering enzyme active sites offers a powerful strategy for tailoring enzyme function.