Kinetics of subtilisin and thiolsubtilisin

Area of Science:

• Biochemistry
• Enzymology

Background:

• Subtilisin is a well-characterized bacterial serine protease.
• Understanding enzyme modifications is crucial for enzyme engineering.

Purpose of the Study:

• To investigate the impact of converting the active site hydroxyl group to a thiol in subtilisin.
• To compare the kinetic and binding properties of thiolsubtilisin with native subtilisin.

Main Methods:

• Enzyme kinetics studies
• Physical techniques for structural analysis
• Substrate binding assays

Main Results:

• Oligopeptide binding is similar for both enzymes.
• Thiolsubtilisin shows increased reactivity with a specific peptide chloromethylketone.
• Thiolsubtilisin exhibits reduced activity and binding towards specific peptide amides/esters and transition state analogs.

Conclusions:

• The OH to SH conversion in subtilisin significantly alters substrate specificity.
• Thiolsubtilisin may have a diminished capacity for transition state stabilization with specific substrates.