Ishii S, Noguchi M, Miyano M, Matsumoto T, Noma M
Life Science Research Laboratory, Japan Tobacco Inc., Kanagawa.
Biochem Biophys Res Commun. 1992 Feb 14;182(3):1482-90. doi: 10.1016/0006-291x(92)91901-2.
Human 5-lipoxygenase contains a non-heme iron essential for its activity. In order to determine which amino acid residues are involved in the iron-binding and the lipoxygenase activity, nine amino acid residues in highly homologous regions among the lipoxygenases were individually replaced by means of site-directed mutagenesis. Mutant 5-lipoxygenases in which His-367 or His-550 was replaced by either Asn or Ala, His-372 by either Asn or Ser, or Glu-376 by Gln were completely devoid of the activity. Though mutants containing an alanine residue instead of His-390 or His-399 lacked the activity, the corresponding asparagine substituted mutants exhibited. The other mutants retained the enzyme activity. These results strongly suggest that His-367, His-372, His-550 and Glu-376 are crucial for 5-lipoxygenase activity and coordinate to the essential iron.
人5-脂氧合酶含有一个对其活性至关重要的非血红素铁。为了确定哪些氨基酸残基参与铁结合和脂氧合酶活性，通过定点诱变分别替换了脂氧合酶中高度同源区域的九个氨基酸残基。其中，将His-367或His-550替换为Asn或Ala、将His-372替换为Asn或Ser、或将Glu-376替换为Gln的突变型5-脂氧合酶完全没有活性。虽然含有丙氨酸残基而非His-390或His-399的突变体缺乏活性，但相应的天冬酰胺取代突变体却有活性。其他突变体保留了酶活性。这些结果有力地表明，His-367、His-372、His-550和Glu-376对5-脂氧合酶活性至关重要，并与必需铁配位。
