PMID:10692590
| Citation |
Kramer, RA, Dekker, N and Egmond, MR (2000) Identification of active site serine and histidine residues in Escherichia coli outer membrane protease OmpT. FEBS Lett. 468:220-4 |
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| Abstract |
Escherichia coli outer membrane protease OmpT has been characterised as a serine protease based on its inhibitor profile, but serine protease consensus sequences are absent. By site-directed mutagenesis we substituted all conserved serines and histidines. Substitution of His(101) and His(212) by Ala, Asn or Gln resulted in variant enzymes with 0.01 and 9-20% residual enzymatic activity towards a fluorogenic pentapeptide substrate, respectively. The mutations S140A and S201A did not decrease activity, while variants S40A and S99A yielded 0.5 and 0.2% residual activities, respectively. When measured with a dipeptide substrate the variant S40A demonstrated full activity, whereas variant S99A displayed at least 500-fold reduced activity. We conclude that Ser(99) and His(212) are essential active site residues. We propose that OmpT is a novel serine protease with Ser(99) as the active site nucleophile and His(212) as general base. |
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| Keywords |
Amino Acid Sequence; Amino Acid Substitution; Binding Sites; Blotting, Western; Cell Membrane/enzymology; Cloning, Molecular; Escherichia coli/enzymology; Escherichia coli/genetics; Histidine; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Structure, Secondary; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism; Serine; Serine Endopeptidases/chemistry; Serine Endopeptidases/genetics; Serine Endopeptidases/metabolism |
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