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Engineering substrate preference in subtilisin: structural and kinetic analysis of a specificity mutant
Biao Ruan1, Viktoriya London, Kathryn E Fisher
1Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, Maryland 20850, USA.
Biochemistry
|May 30, 2008
Summary
Engineering Bacillus subtilisin (Sbt160) improved substrate binding for phenylalanine or tyrosine at P4, but limitations in acylation and product release prevent achieving natural enzyme specificity.
Area of Science:
- Enzymology
- Protein Engineering
- Biochemistry
Background:
- Bacillus subtilisin is a model for engineering substrate specificity.
- Previous engineering efforts show high specificity is challenging to achieve solely through substrate binding.
Purpose of the Study:
- Analyze the structure and kinetics of Sbt160, a subtilisin engineered for P4 phenylalanine/tyrosine preference.
- Identify factors limiting specificity despite improved substrate binding.
Main Methods:
- Structural analysis of inactive Sbt160 with a P4 tyrosine substrate.
- Transient state kinetic analysis using optimal (DFKAM) and suboptimal (DVRAF) sequences.
Main Results:
- Sbt160 shows improved S4 subsite interactions and substrate affinity.
- Acylation rate exceeding substrate dissociation limits discrimination for near-optimal substrates.
- Increased binding energy does not improve transition state stabilization.
- Product release becomes rate-limiting, causing low k(cat) and product inhibition.
Conclusions:
- Achieving high sequence specificity in engineered subtilisins is complex.
- Subtle differences in binding modes for substrates, transition states, and products are difficult to engineer.
- Findings suggest new strategies for engineering highly sequence-selective enzymes.
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