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Related Experiment Videos

Elastase substrate specificity tailored through substrate-assisted catalysis and phage display.

W Dall'Acqua1, C Halin, M L Rodrigues

  • 1Department of Molecular Oncology, Genentech, Inc., 1 DNA Way,South San Francisco, CA 94080, USA.

Protein Engineering
|December 10, 1999
PubMed
Summary
This summary is machine-generated.

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Researchers engineered human neutrophil elastase (HNE) to investigate substrate-assisted catalysis. A modified HNE enzyme demonstrated catalytic activity using a histidine residue within its substrate, opening new possibilities for protease engineering.

Area of Science:

  • Enzymology
  • Protein Engineering
  • Biochemistry

Background:

  • Human neutrophil elastase (HNE) is a serine protease.
  • The catalytic histidine residue is crucial for HNE activity.
  • Investigating alternative catalytic mechanisms is important for enzyme engineering.

Purpose of the Study:

  • To explore substrate-assisted catalysis in HNE by replacing the catalytic histidine with alanine (H57A).
  • To determine if a histidine residue in the substrate can substitute for the missing catalytic histidine.
  • To engineer HNE for novel substrate specificities.

Main Methods:

  • Site-directed mutagenesis was used to create the H57A HNE mutant.
  • Recombinant HNE and H57A were expressed and purified from Pichia pastoris.

Related Experiment Videos

  • Phage display was employed to identify potential histidine-containing substrates.
  • Enzyme kinetics (k(cat)/K(M)) were measured to assess catalytic efficiency.

    • Main Results:

    • The H57A mutant demonstrated catalytic activity with specific histidine-containing peptide substrates.
    • A phage-derived peptide (REHVVY) was cleaved by H57A HNE with significant catalytic efficiency.
    • Substrate preferences at various subsites (P1, P4) were identified.
    • A designed substrate (MEHVVY) showed enhanced cleavage by H57A HNE.

    Conclusions:

    • Substrate-assisted catalysis is feasible for HNE engineering.
    • This strategy enables the creation of highly specific, histidine-dependent proteases.
    • The findings suggest broad applicability to other serine proteases for tailored enzyme design.