Vernet T, Ziomek E, Recktenwald A, Schrag J D, de Montigny C, Tessier D C, Thomas D Y, Cygler M
Biotechnology Research Institute, National Research Council of Canada, Montréal, Québec.
J Biol Chem. 1993 Dec 15;268(35):26212-9.
The three-dimensional structure of lipase II of Geotrichum candidum strain ATCC34614 (GCL II) has provided insights with respect to the nature of the catalytic machinery of lipases. To support these structural observations, we have carried out an analysis of GCL II by mutagenesis. The gene encoding lipase II of Geotrichum candidum strain ATCC34614 (GCL II) was amplified using the polymerase chain reaction, cloned, and sequenced. The intronless lipase gene was expressed and secreted from Saccharomyces cerevisiae at approximately 5 mg/liter of culture. Recombinant GCL II was purified by immunoaffinity chromatography and characterized using a combination of substrates and independent analytical methods. The recombinant enzyme and the enzyme isolated from its natural source have comparable specific activities against triolein of about 1000 mumol of oleic acid released/min/mg of protein. The putative catalytic triad Ser217-His463-Glu354 was probed by site-directed mutagenesis. The substitution of Ser217 by either Cys or Thr and of His463 by Ala led to a complete elimination of the activity against both triolein and tributyrin. Substitution of Glu354 by either Ser, Ala or Gln renders the enzyme inactive and also perturbs the enzyme stability. However, the enzyme with the conservative replacement Glu354 Asp is stable and displays only a small decrease of triolein activity but a 10-fold decrease in activity against tributyrin. There was no appreciable difference in esterase activity between the native, recombinant wild type, and Glu354 Asp mutant. These results confirm that the triad formed by Ser217-Glu354-His463 is essential for catalytic activity. They also show that the active site of GCL II is more tolerant to a conservative change of the carboxylic side chain within the triad than are other hydrolases with similar catalytic triads.
白地霉ATCC34614菌株脂肪酶II（GCL II）的三维结构为了解脂肪酶催化机制的本质提供了线索。为了支持这些结构观察结果，我们通过诱变对GCL II进行了分析。利用聚合酶链反应扩增、克隆并测序了白地霉ATCC34614菌株脂肪酶II（GCL II）的编码基因。无内含子的脂肪酶基因在酿酒酵母中表达并分泌，培养物中浓度约为5 mg/升。重组GCL II通过免疫亲和层析纯化，并使用多种底物和独立分析方法进行表征。重组酶与其天然来源分离的酶对三油酸甘油酯具有相当的比活性，约为每分钟每毫克蛋白质释放1000 μmol油酸。通过定点诱变对推定的催化三联体Ser217-His463-Glu354进行了探究。将Ser217替换为Cys或Thr以及将His463替换为Ala导致对三油酸甘油酯和三丁酸甘油酯的活性完全丧失。将Glu354替换为Ser、Ala或Gln会使酶失活，还会扰乱酶的稳定性。然而，Glu354替换为保守的Asp的酶是稳定的，对三油酸甘油酯活性仅略有下降，但对三丁酸甘油酯的活性下降了10倍。天然型、重组野生型和Glu354 Asp突变体之间的酯酶活性没有明显差异。这些结果证实由Ser217-Glu354-His463形成的三联体对催化活性至关重要。它们还表明，与具有类似催化三联体的其他水解酶相比，GCL II的活性位点对三联体内羧酸侧链的保守变化更具耐受性。
