Key result
Novel probe covalently labels paracaspase active site, revealing hydrophobic preference without detectable amidolytic activity.
Why the study?
The catalytic activity and substrate specificity of the human paracaspase catalytic domain were unclear and required characterization.
Human paracaspase exhibits specific active site labeling at Cys539 but lacks detectable amidolytic activity, suggesting a highly specific or non-canonical catalytic mechanism.
Cautions against assuming canonical protease activity; extends paracaspase biochemistry while leaving open cellular and therapeutic roles.
Human paracaspase has been predicted to be a member of the protein structural fold that encompasses protease clan CD. To determine whether paracaspase has catalytic activity we have expressed the region corresponding to the catalytic domain and used protease activity-based chemical probes to profile the putative active site. A leucine-based acyloxymethyl ketone probe that covalently labels cysteine proteases discloses a hydrophobic P 1 preference in the putative active site. The probe covalently labels Cys539, which is not the predicted catalytic site based on structural and sequence comparisons with other clan CD proteases. Using a combinatorial peptide substrate library approach we have been unable to detect amidolytic activity of paracaspase, implying that if it is a protease it must be very specific. We suggest a switch in the use of catalytic residues to generate an enzyme overlapping the canonical clan CD protease active site.
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Snipas et al. (2004) studied this question. Protease activity-based chemical probes was evaluated on Catalytic activity and active site profiling. A leucine-based acyloxymethyl ketone probe covalently labeled Cys539 in the putative active site of human paracaspase, revealing a hydrophobic P1 preference without detectable amidolytic activity.
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