Journal
FEBS LETTERS
Volume 468, Issue 2-3, Pages 220-224Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/S0014-5793(00)01231-X
Keywords
active site; serine protease; OmpT outer membrane protein; Escherichia coli
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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,vith 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. (C) 2000 Federation of European Biochemical Societies.
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