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Engineering the primary substrate specificity of Streptomyces griseus trypsin
Michael J Page1, Sui-Lam Wong, Jeff Hewitt
1Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Biochemistry
|July 30, 2003
Summary
Engineered Streptomyces griseus trypsin (SGT) mutants enhance substrate specificity. The T190P mutant shows increased preference for Arg over Lys at the P1 site with minimal impact on catalytic efficiency.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Streptomyces griseus trypsin (SGT) serves as a model for developing serine proteases with improved substrate specificity.
- Recombinant SGT was successfully produced in Bacillus subtilis, purified, and characterized.
Purpose of the Study:
- To engineer SGT mutants with altered substrate specificity, focusing on the S1 binding pocket.
- To investigate the structural and enzymatic consequences of mutations in the S1 pocket.
Main Methods:
- Site-directed mutagenesis was used to create four SGT mutants (T190A, T190P, T190S, T190V).
- Recombinant proteins were expressed, purified, and enzymatically characterized.
- High-resolution crystal structures of wild-type SGT and the T190P mutant complexed with benzamidine were determined.
Main Results:
- The T190P mutant exhibited a significant shift in P1 substrate specificity, favoring arginine over lysine.
- This specificity enhancement occurred with only a 25% reduction in catalytic activity for arginine-containing substrates.
- Crystal structures revealed the molecular basis for the altered substrate binding.
Conclusions:
- The T190P mutation effectively enhances SGT's P1 specificity with minimal loss of catalytic efficiency.
- This engineered mutant is a promising scaffold for designing serine proteases with tailored substrate specificities for the S2-S4 pockets.