Isolation and characterization of a lysine-specific protease from Pseudomonas aeruginosa.

We report here a procedure which results in the purification of an extracellular protease (designated Ps-1) from Pseudomonas aeruginoaa.This enzyme cleaves fibrinogen so that the modified molecules form microcrystals and large single crystals.Precise knowledge of the Ps-1 cleavage sites is essential for the interpretation of the structural information provided by these crystals (Weisel, J. W., Stauffacher, C. V., Bullitt, E., and Cohen, C. (1985) Science 230, 1388-1391).Ps-1 is a single-chain polypeptide of M, 30,000 which appears to function as a monomer.The pH optimum is 8-9.The activity of the protease is not decreased by inhibitors of thiol, carboxyl, or metallo proteases; the abolishment of activity by N"-p-tosyI-Llysine chloromethyl ketone and the partial inhibition obtained with serine-reactive inhibitors suggests that Ps-1 may be a serine protease with an unusual activesite conformation.Studies with synthetic peptide substrates show that Ps-1 exhibits one of the most restricted specificities known for an endoproteinase: only peptide, ester, and amide bonds containing the carbonyl group of lysine are hydrolyzed.The limited specificity of Ps-1 should make it useful for other applications requiring the selective cleavage of proteins, such as sequence analysis and the isolation of domains.* Boehringer-Mannheim Biochemicals, information supplied with the enzyme preparation.