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Related Experiment Videos

Converting trypsin to chymotrypsin: ground-state binding does not determine substrate specificity

L Hedstrom1, S Farr-Jones, C A Kettner

  • 1Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.

Biochemistry
|July 26, 1994
PubMed
Summary
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Altering surface loops in rat trypsin II created chymotrypsin-like activity. This modification enhances transition state stabilization for chymotrypsin substrates, impacting enzyme specificity.

Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Engineering

Background:

  • Rat trypsin II normally exhibits trypsin-like substrate specificity.
  • Chymotrypsin possesses distinct substrate specificity due to its unique structural features.
  • Enzyme specificity is crucial for biological function and drug development.

Purpose of the Study:

  • To investigate the role of specific surface loops in determining protease substrate specificity.
  • To engineer rat trypsin II to exhibit chymotrypsin-like substrate specificity.
  • To elucidate the mechanisms by which surface loops influence catalytic activity and substrate binding.

Main Methods:

  • Site-directed mutagenesis to exchange surface loops between rat trypsin II and chymotrypsin.
  • Enzyme kinetics assays using specific substrates and inhibitors (chloromethylketones and boronic acids).

Related Experiment Videos

  • Analysis of substrate hydrolysis rates (kcat/Km) and inhibition constants (Ki).
  • Proflavin binding assays to assess substrate-binding pocket integrity.

    • Main Results:

    • Mutant rat trypsin II (Tr-->Ch[S1+L1+L2] and Tr-->Ch[S1+L1+L2+Y172W]) displayed chymotrypsin-like substrate specificity.
    • The exchange of surface loops correlated with improved catalytic efficiency (kcat/Km) for chymotrypsin substrates.
    • Surface loops were found to stabilize the transition state by optimizing substrate orientation.
    • Mutations significantly altered substrate-binding pocket affinity and structure, as evidenced by proflavin binding.

    Conclusions:

    • Enzyme specificity is primarily determined by the chemical steps of the catalytic reaction, not solely by substrate binding.
    • Surface loop structure plays a critical role in modulating protease activity and specificity.
    • Protein engineering by loop exchange is a viable strategy to alter enzyme function.