Site‐directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys‐pocket, and Glu347 and Arg350 of the EXXR motif

FEBS Letters

Hatae, Toshihisa²  Inoue, Hiroyasu²  Yabuki, Tomoko²  Ullrich, Volker¹  Yokoyama, Chieko²  Tanabe, Ta-i²  Hara, Shuntaro² 

[1] Faculty of Biology, University of Konstanz, P.O. Box 5560, D-78434 Konstanz, Germany;Department of Pharmacology, National Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Osaka 565, Japan

关键词: Prostacyclin synthase;    Prostaglandin I2;    Site-directed mutagenesis;    Cytochrome P450;    PG;    prostaglandin;    PGI2;    prostacyclin;    PCR;    polymerase chain reaction;    DTT;    dithiothreitol;    BHT;    butylated hydroxytoluene;    PVDF;    polyvinylidene difluoride;    KPB;    potassium phosphate buffer;   

DOI  :  10.1016/0014-5793(96)00600-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The possible active site Cys⁴⁴¹ in the Cys-pocket and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif of the human prostacyclin synthase, which catalyzes the conversion of prostaglandin H₂ to prostacyclin, were subjected to site-directed mutagenesis in order to understand the role of these residues in expressing the enzymatic activity. Five expression vectors encoding the mutant enzymes with a single replacement, Cys⁴⁴¹Ala, Cys⁴⁴¹Ser, Cys⁴⁴¹His, Glu³⁴⁷Ala and Arg³⁵⁰Ala, as well as the wild-type enzyme were expressed in 293 cells. The microsomal fraction of the cells expressing the wild-type enzyme showed a specific activity of 96 nmol 6-keto-PGF_1α/min per mg protein. All of the mutant enzymes examined showed no detectable enzyme activity, although immunoblot analysis demonstrated that levels of all the expressed mutant enzymes were similar to that of the wild-type enzyme. These results indicated that the Cys⁴⁴¹ in the Cyspocket, and Glu³⁴⁷ and Arg³⁵⁰ of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.

【 授权许可】

Unknown