Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site

FEBS Letters

de Roon, Gerard Jan¹  Kramer, R.Arjen¹  Dekker, Niek¹  Egmond, Maarten R.¹  Vandeputte-Rutten, Lucy²  Gros, Piet² 

[1] Department of Enzymology and Protein Engineering, Center for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands;Department of Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

关键词: Active site;    Asp/His dyad;    Protease;    OmpT;    Outer membrane protein;    Abz;    o-aminobenzoyl;    Dap(dnp);    N-β-dinitrophenyl-L-diaminopropionic acid;    DFP;    diisopropylfluorophosphate;    PMSF;    phenylmethylsulfonyl fluoride;    Triton X-100;    polyethyleneglycol tert-octylphenyl ether;    Tween 20;    polyoxyethylene sorbitanmonolaurate;   

DOI  :  10.1016/S0014-5793(01)02863-0

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 previously been classified as a serine protease with Ser⁹⁹ and His²¹² as active site residues. The recently solved X-ray structure of the enzyme was inconsistent with this classification, and the involvement of a nucleophilic water molecule was proposed. Here, we substituted all conserved aspartate and glutamate residues by alanines and measured the residual enzymatic activities of the variants. Our results support the involvement of a nucleophilic water molecule that is activated by the Asp²¹⁰/His²¹² catalytic dyad. Activity is also strongly dependent on Asp⁸³ and Asp⁸⁵. Both may function in binding of the water molecule and/or oxyanion stabilization. The proposed mechanism implies a novel proteolytic catalytic site.

【 授权许可】

Unknown