FEBS Letters | |
Identification of active site serine and histidine residues in Escherichia coli outer membrane protease OmpT | |
Egmond, Maarten R1  Kramer, R.Arjen1  Dekker, Niek1  | |
[1] Department of Enzymology and Protein Engineering, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands | |
关键词: Active site; Serine protease; OmpT; Outer membrane protein; Escherichia coli; Abz; o-aminobenzoyl; DFP; diisopropylfluorophosphate; GAMAP; goat anti-mouse IgG alkaline phosphatase conjugate; IAA; indole-3-acetic acid; PMSF; phenylmethylsulfonyl fluoride; pNA; p-nitroaniline; Triton X-100; polyethyleneglycol tert-octylphenyl ether; Tween 20; polyoxyethylene sorbitanmonolaurate; Tyr(NO2); 3-nitrotyrosine; | |
DOI : 10.1016/S0014-5793(00)01231-X | |
学科分类:生物化学/生物物理 | |
来源: John Wiley & Sons Ltd. | |
【 摘 要 】
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 His101 and His212 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 Ser99 and His212 are essential active site residues. We propose that OmpT is a novel serine protease with Ser99 as the active site nucleophile and His212 as general base.
【 授权许可】
Unknown
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