Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides

Area of Science:

• Biochemistry
• Enzymology
• Proteolysis

Background:

• Cathepsin B is a cysteine protease with both endopeptidase and carboxydipeptidase activities.
• Understanding the substrate specificity of cathepsin B is crucial for its role in various physiological and pathological processes.

Purpose of the Study:

• To elucidate the substrate specificity of the carboxydipeptidase activity of cathepsin B at its S1, S2, S1', and S2' subsites.
• To develop efficient and sensitive substrates for analyzing cathepsin B carboxydipeptidase activity.

Main Methods:

• Synthesis and assay of internally quenched fluorescent peptides based on Dnp-GFRFW-OH.
• Systematic substitution of amino acids at different positions (P1, P2, P1', P2') to probe subsite preferences.
• Utilized substrates with o-aminobenzoic acid and 3-Dnp-(2,3-diaminopropionic acid) or epsilon-amino-Dnp-Lys as donor-receptor pairs.

Main Results:

• Subsite S1 preferentially accepted basic amino acids, but showed lower Km values for phenylalanine and aliphatic side-chain amino acids.
• Subsite S2 exhibited a preference for aromatic residues, with lysine at P2 being better accommodated than arginine.
• Subsite S1' was identified as a hydrophobic subsite.
• Subsite S2' showed a specific preference for phenylalanine or tryptophan residues.

Conclusions:

• Detailed characterization of cathepsin B carboxydipeptidase subsite specificity.
• Identification of key amino acid preferences at each binding pocket (S1, S2, S1', S2').
• Provides insights into substrate recognition mechanisms for cathepsin B's carboxydipeptidase function.