Identification of active site serine and histidine residues in Escherichiacoli outer membrane protease OmpT

Escherichia coli outer membrane protease OmpT has been characterised as a s erine protease based on its inhibitor profile, but serine protease consensu s sequences are absent. By site-directed mutagenesis we substituted all con served 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 enzym atic activity towards a fluorogenic pentapeptide substrate, respectively. T he mutations S140A and S201A did not decrease activity, while variants S40A and S99A yielded 0.5 and 0.2% residual activities, respectively. When meas ured,vith a dipeptide substrate the variant S40A demonstrated full activity ., whereas variant S99A displayed at least 500-fold reduced activity. We co nclude that Ser(99) and His(212) are essential active site residues. We pro pose that OmpT is a novel serine protease with Ser(99) as the active site n ucleophile and His(212) as general base. (C) 2000 Federation of European Bi ochemical Societies.