Conversion of trypsin to a functional threonine protease

Area of Science:

• Enzymology
• Protein engineering
• Biochemistry

Background:

• Serine proteases utilize a catalytic serine residue, rarely employing the similar threonine residue.
• Steric hindrance from threonine's beta-carbon methyl group and disulfide bridges typically inhibits its function in protease active sites.

Purpose of the Study:

• To engineer a functional threonine protease by modifying the classic serine protease, trypsin.
• To investigate the structural and functional implications of substituting serine with threonine at the catalytic site.

Main Methods:

• Structural modeling to predict steric interactions.
• Site-directed mutagenesis to substitute serine 195 with threonine.
• Truncation of disulfide bridge-forming residues (cysteines 42 and 58).
• Enzymatic activity assays (kcat/KM) and substrate specificity analysis.

Main Results:

• A Ser 195 --> Thr trypsin variant was inactive due to steric clashes.
• Simultaneous truncation of Cys42 and Cys58 with Ser 195 --> Thr substitution yielded a functional threonine protease.
• Mutant threonine proteases showed reduced, but significantly enhanced, activity compared to the single Ser 195 --> Thr variant.
• Substrate specificity was retained, while thermostability decreased but was partially restored by threonine.

Conclusions:

• Overcoming steric hindrance by removing disulfide bridges enables the creation of functional threonine proteases.
• This work expands the catalytic repertoire of proteases beyond serine.
• Threonine can serve as a functional catalytic residue in engineered proteases.