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Engineered metal regulation of trypsin specificity
W S Willett1, S A Gillmor, J J Perona
1Department of Pharmaceutical Chemistry, University of California at San Francisco 94143-0446.
Biochemistry
|February 21, 1995
Summary
Researchers engineered histidine substrate specificity into trypsin using metal ions (Ni2+, Zn2+). This demonstrates how metal cofactors can precisely modulate enzyme specificity, enabling new catalytic functions.
Area of Science:
- Enzymology
- Protein Engineering
- Biochemistry
Background:
- Trypsin typically exhibits broad specificity, cleaving after basic residues.
- Modifying enzyme specificity is crucial for developing targeted biocatalysts.
- Metal ions can influence protein structure and function.
Purpose of the Study:
- To engineer histidine substrate specificity into trypsin.
- To investigate the role of metal ions in modulating enzyme specificity.
- To explore the feasibility of altering trypsin's P2' and P1 substrate binding sites.
Main Methods:
- Site-directed mutagenesis was used to create specific amino acid substitutions in trypsin (N143H/E151H and D189H).
- Metal binding sites for Ni2+ and Zn2+ were engineered into the enzyme.
- Kinetic assays and crystallographic analysis were employed to evaluate substrate hydrolysis and structural changes.
Main Results:
- The N143H/E151H trypsin variant showed exclusive hydrolysis of P2'-His-containing peptides in the presence of Ni2+ or Zn2+.
- This engineered specificity was achieved with high catalytic efficiency, even for normally disfavored cleavage sites.
- A single-site mutation (D189H) in the S1 pocket was unsuccessful due to structural perturbations, indicating limitations in local pocket deformation.
Conclusions:
- Metal cofactors can be effectively used to design and modulate enzyme specificity.
- Engineering specificity at the P2' position of trypsin is feasible using metal-binding sites.
- Specificity modification at the trypsin S1 site is challenging due to the pocket's inability to deform locally, necessitating strategies utilizing extended subsites.