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Functional interaction among catalytic residues in subtilisin BPN'.
1Department of Protein Engineering, Genentech Inc., South San Francisco, California 94080.
Proteins
|January 1, 1990
Summary
Mutating subtilisin BPN's Asn-155 residue significantly reduces enzyme activity, but its catalytic role is dependent on Ser-221. This study investigates residual enzyme activity and inhibitor binding in engineered subtilisin variants.
Area of Science:
- Biochemistry
- Enzymology
- Protein Engineering
Background:
- Subtilisin BPN' is a serine protease with a catalytic triad (Ser-221, His-64, Asp-32).
- Residual activity in catalytic triad variants suggests contributions from other residues, like Asn-155.
- Asn-155 stabilizes the oxyanion transition state in wild-type subtilisin.
Purpose of the Study:
- To investigate the role of Asn-155 in subtilisin BPN's catalytic activity.
- To characterize the impact of Asn-155 mutations on enzyme kinetics and inhibitor binding.
- To understand the interplay between Asn-155 and Ser-221 in catalysis.
Main Methods:
- Site-directed mutagenesis to create Asn-155 Gly (N155G) and Ser-221 Ala (S221A) variants.
- Enzyme kinetic assays using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPF-pna) substrate.
- Inhibition studies using turkey ovomucoid inhibitor (OMTKY3).
Main Results:
- N155G mutation decreased turnover number (kcat) by 150-fold with minimal change in Michaelis constant (KM).
- The double mutant N155G:S221A showed a 5-fold increase in kcat compared to S221A alone.
- Mutations weakened subtilisin-OMTKY3 binding, with N155G and S221A reducing binding affinity by 1.8 and 2.0 kcal/mol, respectively.
Conclusions:
- The catalytic function of Asn-155 in subtilisin is dependent on the presence of Ser-221.
- Asn-155 contributes to transition state stabilization, evidenced by reduced activity and altered inhibitor binding.
- The residual activity of engineered subtilisin variants is biologically relevant and can be inhibited.