Zhang Y Y, Lind B, Rådmark O, Samuelsson B
Department of Physiological Chemistry, Karolinska Institute, Stockholm, Sweden.
J Biol Chem. 1993 Feb 5;268(4):2535-41.
Recombinant human 5-lipoxygenase was expressed in Escherichia coli and purified to more than 95% homogeneity by ammonium sulfate precipitation and agarose-ATP column chromatography. The specific activity of the purified enzyme was 21-28 mumol/mg, as assessed by the generation of 5-hydro(pero)xyeicosatetraenoic acid. The iron content was analyzed by graphite furnace atomic absorption spectrophotometry for six preparations of the enzyme. The average value of the iron content was 0.86 mol/mol (iron/protein) with a range of 0.74-1.15 mol/mol. All lipoxygenases that have been sequenced contain 6 conserved histidine residues. Mutants of 5-lipoxygenase, with substitutions of these 6 conserved histidines, were purified and analyzed. Mutants H372Q and H550Q had no detectable enzyme activity and were also practically devoid of iron. Three mutants regarding His367 (H367Q, H367N, and H367S) were all inactive but had partial iron contents (0.5, 0.2, and 0.5 mol/mol, respectively). Finally, the mutated proteins H362Q, H390Q, and H399Q displayed reduced enzyme activity but contained similar amounts of iron as non-mutated 5-lipoxygenase. We conclude that histidines 372 and 550 constitute two of the iron ligands in 5-lipoxygenase. Also His367 is necessary for the enzyme activity, but this residue is not crucial for binding of iron.
重组人5-脂氧合酶在大肠杆菌中表达，并通过硫酸铵沉淀和琼脂糖-ATP柱色谱法纯化至纯度超过95%。通过5-氢（过）氧二十碳四烯酸的生成来评估，纯化酶的比活性为21-28 μmol/mg。通过石墨炉原子吸收分光光度法对该酶的六种制剂进行铁含量分析。铁含量的平均值为0.86 mol/mol（铁/蛋白质），范围为0.74-1.15 mol/mol。所有已测序的脂氧合酶都含有6个保守的组氨酸残基。对5-脂氧合酶的这些6个保守组氨酸进行替换的突变体进行了纯化和分析。突变体H372Q和H550Q没有可检测到的酶活性，并且实际上也不含铁。关于His367的三个突变体（H367Q、H367N和H367S）均无活性，但铁含量部分保留（分别为0.5、0.2和0.5 mol/mol）。最后，突变蛋白H362Q、H390Q和H399Q显示出降低的酶活性，但所含铁量与未突变的5-脂氧合酶相似。我们得出结论，组氨酸372和550构成5-脂氧合酶中的两个铁配体。His367对酶活性也是必需的，但该残基对铁的结合并不关键。
