Defining the substrate specificity of mouse cathepsin P

Area of Science:

• Biochemistry
• Enzymology

Background:

• Cathepsin P is a newly identified cysteine protease from the placenta.
• It shares structural similarities with cathepsin L, a broad-specificity enzyme.

Purpose of the Study:

• To investigate the substrate specificity of recombinant mouse cathepsin P.
• To map the enzyme's S(3) to S(2)(') subsites.

Main Methods:

• Utilized fluorescence resonance energy transfer (FRET) peptides for substrate analysis.
• Systematically modified peptide sequences to probe enzyme-substrate interactions.
• Assessed the effect of kosmotropic salts (e.g., Na(2)SO(4)) on enzyme activity.
• Employed circular dichroism and size exclusion chromatography to evaluate structural changes.

Main Results:

• Cathepsin P exhibits a strong preference for hydrophobic residues at the S(1), S(2), S(1)('), and S(2)(') subsites.
• The S(2) subsite is particularly selective for hydrophobic aliphatic amino acids.
• The S(3) subsite demonstrates broad specificity, accommodating charged and hydrophobic residues.
• Kosmotropic salts significantly influenced cathepsin P activity, likely by disrupting ionic interactions.
• A high-efficiency substrate was identified with a k(cat)/K(m) nearly two orders of magnitude greater than the parent compound.

Conclusions:

• Cathepsin P possesses a distinct and restricted catalytic specificity compared to other mammalian cathepsins.
• Understanding these specificity requirements is crucial for its functional characterization and potential applications.