Identification of the active site residues of Pseudomonas aeruginosa protease IV. Importance of enzyme activity in autoprocessing and activation.
Protease IV is a lysine-specific endoprotease produced by Pseudomonas aeruginosa whose activity has been correlated with corneal virulence. Comparison of the protease IV amino acid sequence to other bacterial proteases suggested that amino acids His-72, Asp-122, and Ser-198 could form a catalytic triad that is critical for protease IV activity. To test this possibility, site-directed mutations by alanine substitution were introduced into six selected residues including the predicted triad and identical residues located close to the triad. Mutations at any of the amino acids of the predicted catalytic triad or Ser-197 caused a loss of enzymatic activity and absence of the mature form of protease IV. In contrast, mutations at His-116 or Ser-200 resulted in normal processing into the enzymatically active mature form. A purified proenzyme that accumulated in the His-72 mutant was shown in vitro to be susceptible to cleavage by protease IV purified from P. aeruginosa. Furthermore, similarities of protease IV to the lysine-specific endoprotease of Achromobacter lyticus suggested three possible disulfide bonds in protease IV. These results identify the catalytic triad of protease IV, demonstrate that autodigestion is essential for the processing of protease IV into a mature protease, and predict sites essential to enzyme conformation.
Traidej
Mullika
M
Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center, New Orleans 70112, USA.
Marquart
Mary E
ME
Caballero
Armando R
AR
Thibodeaux
Brett A
BA
O'Callaghan
Richard J
RJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry
D004220:Disulfides; D012694:Serine; D010447:Peptide Hydrolases; C030732:protease IV