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 aeruginosa. 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 Mr 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 alpha-p-tosyl-L-lysine chloromethyl ketone and the partial inhibition obtained with serine-reactive inhibitors suggests that Ps-1 may be a serine protease with an unusual active-site 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.
D000596:Amino Acids / Q000032:analysis; D002850:Chromatography, Gel; D003460:Crystallization; D005340:Fibrinogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
D000596:Amino Acids; D005340:Fibrinogen; D010447:Peptide Hydrolases; D008239:Lysine