Site-directed mutagenesis of human prostacyclin synthase: Alteration of Cys441 of the Cys-pocket, and Glu347 and Arg350 of the EXXR motif.
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, Cys441 Ala, Cys441 Ser, Cys441 His, Glu347 Ala and Arg350 Ala, 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-PGF1alpha/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 Cys-pocket, and Glu347 and Arg350 of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.
Hatae
T
T
Department of Pharmacology, National Cardiovascular Center Research Institute, Osaka, Japan.
Hara
S
S
Yokoyama
C
C
Yabuki
T
T
Inoue
H
H
Ullrich
V
V
Tanabe
T
T
D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D002855:Chromatography, Thin Layer; D017124:Conserved Sequence; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D044262:Prostaglandin H2; D011463:Prostaglandins H / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment
D017931:DNA Primers; D011463:Prostaglandins H; D011994:Recombinant Proteins; D044262:Prostaglandin H2; D003577:Cytochrome P-450 Enzyme System; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; C022461:prostacyclin synthetase