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Substrate specificity of trypsin investigated by using a genetic selection
L B Evnin1, J R Vásquez, C S Craik
1Department of Biochemistry, University of California, San Francisco 94143.
Summary
Investigating rat anionic trypsin, this study reveals that a negative charge at positions 189 or 190 is crucial for catalytic activity. Amino acid variations at position 190 modulate specificity for arginine and lysine substrates.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Rat anionic trypsin is a key digestive enzyme.
- Understanding its substrate specificity is vital for enzyme engineering and drug development.
Purpose of the Study:
- To elucidate the structural determinants of rat anionic trypsin's primary substrate specificity.
- To identify key amino acid residues influencing catalytic activity and substrate preference.
Main Methods:
- Oligonucleotide-directed mutagenesis was employed to create a library of trypsin variants.
- A high-dynamic-range genetic selection was used to screen 90,000 transformants.
- Kinetic characterization of purified wild-type and mutant trypsins on arginyl and lysyl peptide substrates.
Main Results:
- A negative charge at amino acid positions 189 or 190 was essential for high-level catalytic activity.
- Mutations at position 190 modulated specificity for arginine and lysine substrates, while position 189 favored aspartic acid.
- Alternative amino acid arrangements in the substrate binding pocket sustained efficient catalysis.
Conclusions:
- The study identifies critical residues (189 and 190) in the trypsin substrate binding pocket that dictate substrate specificity.
- A negative charge at these positions is essential for efficient catalysis.
- Findings provide insights into the structure-function relationships of trypsin homologs.