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

FEBS Letters

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