Substrate specificity of the streptococcal cysteine protease

Area of Science:

• Biochemistry
• Microbiology
• Enzymology

Background:

• Streptococcal pyrogenic exotoxin B (SpeB) is crucial in Streptococcus pyogenes pathogenesis.
• SpeB is the zymogen form of streptococcal cysteine protease (SCP), with limited known substrate specificity.
• Understanding SCP's substrate specificity is key to elucidating its role in infections.

Purpose of the Study:

• To investigate the substrate specificity of streptococcal cysteine protease (SCP).
• To design and utilize novel fluorescent substrates for activity assays.
• To compare SCP's specificity with other cysteine proteases, like those in the papain family.

Main Methods:

• Design of internally quenched fluorescent substrates based on SpeB autoprocessing sites.
• Enzyme kinetics assays using designed substrates to determine activity (kcat/KM).
• High-pressure liquid chromatography (HPLC) and mass spectrometry (MS) to confirm cleavage sites.

Main Results:

• Optimal SCP substrates feature specific amino acid sequences (e.g., AIK) in the P(3)-P(1) positions.
• The pentamer substrate 3-aminobenzoic acid-AIKAG-3-nitrotyrosine exhibited the highest activity, spanning S(3) to S(2)' subsites.
• SCP demonstrates endopeptidase activity, with a preference for hydrophobic residues at the S(2) subsite.

Conclusions:

• SCP exhibits significant endopeptidase activity with defined substrate preferences.
• The determined substrate specificity aligns with that of papain-family cysteine proteases.
• SCP's specificity profile likely applies to processing both mammalian and streptococcal protein targets.