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Electrostatic complementarity within the substrate-binding pocket of trypsin.

L Gráf1, A Jancsó, L Szilágyi

  • 1L. Eotvos University, Biochemistry Department, Budapest, Hungary.

Proceedings of the National Academy of Sciences of the United States of America
|July 1, 1988
PubMed
Summary
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Replacing aspartic acid with serine in trypsin significantly alters substrate specificity. The mutant enzyme shows drastically reduced activity on preferred substrates but increased activity on chymotrypsin-type substrates, highlighting the role of this residue in enzyme-substrate interactions.

Area of Science:

  • Biochemistry
  • Enzymology
  • Protein Engineering

Background:

  • Trypsin's substrate specificity is crucial for its biological function.
  • The aspartic residue at position 189 (Asp-189) in trypsin's substrate-binding pocket is key for its preference for basic residues.
  • Chymotrypsin, which has a serine at this position, prefers hydrophobic residues.

Purpose of the Study:

  • To investigate the role of Asp-189 in trypsin's substrate specificity.
  • To engineer trypsin to exhibit chymotrypsin-like substrate preference.

Main Methods:

  • Site-directed mutagenesis was used to replace Asp-189 with serine in trypsinogen.
  • Wild-type and mutant trypsinogens were expressed in E. coli, purified, and activated.
  • Enzyme kinetics were assessed using fluorogenic tetrapeptide substrates with varying C-terminal residues (Lys, Arg, Tyr, Phe, Leu, Trp).

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Main Results:

  • The [Ser189]trypsin mutant showed a 5-log-unit decrease in activity against lysyl and arginyl substrates compared to wild-type trypsin.
  • The mutant enzyme exhibited a 10-50 fold increase in activity towards chymotrypsin-type substrates (Tyr, Phe, Leu, Trp).
  • The activity of [Ser189]trypsin on lysyl substrates was pH-dependent, favoring unprotonated lysine at higher pH.

Conclusions:

  • The Asp-189 residue is critical for trypsin's specificity towards basic substrates through electrostatic interactions.
  • Replacing Asp-189 with serine converts trypsin into an enzyme with broader specificity, including chymotrypsin-like activity.
  • Transition-state energy calculations suggest that electrostatic and hydrogen-bond interactions involving residue 189 significantly influence enzyme-substrate binding affinity.