C-TERMINAL SPECIFIC PROTEIN-DEGRADATION - ACTIVITY AND SUBSTRATE-SPECIFICITY OF THE TSP PROTEASE

The activity of Tsp, a periplasmic endoprotease of Escherichia coli, h as been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction. Tsp efficiently c leaves substrates that have apolar residues and a free alpha-carboxyla te at the C-terminus. Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity. In additi on to preferences for residues at the C-terminus and cleavage sites, T sp displays a preference for substrates that are not stably folded: un stable variants of Arc repressor are better substrates than a hypersta ble mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate. These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C-terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bo nds.