Nonessential active site residues modulate selenosubtilisin's kinetic mechanism

Area of Science:

• Biochemistry
• Enzyme Engineering
• Biocatalysis

Background:

• Selenosubtilisin is a semisynthetic enzyme mimicking glutathione peroxidase.
• It catalyzes hydroperoxide reduction using 3-carboxy-4-nitrobenzenethiol.
• Kinetic properties vary significantly between different selenosubtilisin variants.

Purpose of the Study:

• To investigate the kinetic properties and mechanisms of selenosubtilisins derived from different subtilisin templates.
• To identify the role of specific amino acid substitutions in the active site on enzyme kinetics and efficiency.
• To explore the potential for controlling selenosubtilisin mechanism through targeted mutagenesis.

Main Methods:

• Chemical modification of subtilisin to produce selenosubtilisin.
• Kinetic analysis of selenosubtilisin BPN' and its variants.
• Site-directed mutagenesis of active site residues (S1 and S1' binding sites).
• Comparison of kinetic properties between different selenosubtilisin variants and mutants.

Main Results:

• Selenosubtilisin BPN' exhibits lower activity and sequential kinetics compared to selenosubtilisin Carlsberg.
• Kinetic mechanism and efficiency differences are attributed to amino acid substitutions in the S1 and S1' binding sites.
• A triple mutant (E156S/G169A/Y217L) of BPN' closely approximates the properties of selenosubtilisin Carlsberg.

Conclusions:

• The kinetic mechanism of selenosubtilisin is tunable via limited mutagenesis of active site residues.
• Mutations in S1 and S1' binding sites significantly impact enzyme kinetics and efficiency.
• This study provides a basis for engineering selenosubtilisin variants with desired kinetic properties for biocatalysis.