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Related Experiment Videos

Delocalizing trypsin specificity with metal activation

W S Willett1, L S Brinen, R J Fletterick

  • 1Department of Pharmaceutical Chemistry, University of California at San Francisco 94143-0446, USA.

Biochemistry
|May 14, 1996
PubMed
Summary
This summary is machine-generated.

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Engineered trypsin variants show altered substrate specificity, with mutations enabling metal-assisted catalysis. This research demonstrates delocalized protease specificity through engineered subsites, enhancing catalytic efficiency for specific peptide substrates.

Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Engineering

Background:

  • Trypsin's substrate specificity is primarily determined by the P1 pocket, which tightly binds arginine or lysine residues.
  • Extended subsite interactions contribute to catalysis, but P1 pocket binding dominates specificity.
  • Previous studies suggested mutations could alter trypsin's specificity.

Purpose of the Study:

  • To engineer trypsin variants with altered substrate specificity, particularly for tyrosine at P1 and histidine at P2'.
  • To investigate the role of metal ions in the catalytic activity of engineered trypsin.
  • To demonstrate the concept of delocalizing protease specificity through engineered subsites.

Main Methods:

  • Site-directed mutagenesis was used to create trypsin variants (N143H, E151H, D189S).

Related Experiment Videos

  • Enzyme kinetics were measured for wild-type and mutant trypsin with specific peptide substrates in the presence and absence of transition metals (Ni, Zn, Cu).
  • Analysis focused on changes in catalytic efficiency and substrate binding contributions.

    • Main Results:

    • The triple mutant trypsin (N143H, E151H, D189S) showed increased activity towards a tyrosine-containing peptide (AGPYAHSS) in the presence of nickel (25-fold) and zinc (150-fold).
    • This mutant also exhibited enhanced activity towards an arginine-containing peptide (YLVGPRGHFYDA) with copper (30-fold), nickel (70-fold), and zinc (350-fold).
    • Metal-assisted catalysis significantly boosted the activity of the engineered trypsin variants.

    Conclusions:

    • Engineering multiple substrate binding subsites in trypsin can effectively delocalize protease specificity.
    • Altered substrate binding contributions from engineered subsites, in conjunction with metal ions, can enhance catalytic efficiency.
    • This approach offers a strategy for tailoring protease activity and specificity for targeted applications.