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Trypsin: a case study in the structural determinants of enzyme specificity

L Hedstrom1

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

Biological Chemistry
|July 1, 1996
PubMed
Summary

Altering trypsin

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Protein science : a publication of the Protein Society·2001

Area of Science:

  • Enzymology
  • Structural Biology
  • Protein Engineering

Background:

  • Trypsin and chymotrypsin are serine proteases with similar structures but distinct substrate specificities.
  • Trypsin cleaves after basic residues (Lys/Arg), while chymotrypsin targets large hydrophobic residues.

Purpose of the Study:

  • To investigate the structural basis of substrate specificity in trypsin and chymotrypsin.
  • To determine if altering the S1 site alone confers chymotrypsin-like activity to trypsin.

Main Methods:

  • Site-directed mutagenesis of trypsin to mimic chymotrypsin's S1 site residues.
  • Introduction of surface loop substitutions and specific residue changes (Tyr172 to Trp).
  • Enzyme kinetics assays and X-ray crystallography of mutant enzymes.

Main Results:

  • S1 site mutations alone did not confer chymotrypsin-like activity.
  • Substituting surface loops and modifying Tyr172 restored chymotrypsin-like catalytic rates but impaired substrate binding.
  • X-ray structures revealed loop disorder, stabilized by the Tyr172 to Trp substitution.

Conclusions:

  • Enzyme substrate specificity arises from a network of interactions extending beyond the primary substrate-binding site.
  • Surface loop conformation and specific residue interactions outside the S1 pocket are crucial for defining protease activity.

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