Moreau A, Roberge M, Manin C, Shareck F, Kluepfel D, Morosoli R
Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Laval, Québec, Canada.
Biochem J. 1994 Aug 15;302 ( Pt 1)(Pt 1):291-5. doi: 10.1042/bj3020291.
On the basis of similarities between known xylanase sequences of the F family, three invariant acidic residues of xylanase A from Streptomyces lividans were investigated. Site-directed-mutagenesis experiments were carried out in Escherichia coli after engineering the xylanase A gene to allow its expression. Replacement of Glu-128 or Glu-236 by their isosteric form (Gln) completely abolished enzyme activity with xylan and p-nitrophenyl beta-D-cellobioside, indicating that the two substrates are hydrolysed at the same site. These two amino acids probably represent the catalytic residues. Immunological studies, which showed that the two mutants retained the same epitopes, indicate that the lack of activity is the result of the mutation rather than misfolding of the protein. Mutation D124E did not affect the kinetic parameters with xylan as substrate, but D124N reduced the Km 16-fold and the Vmax. 14-fold when compared with the wild-type enzyme. The mutations had a more pronounced effect with p-nitrophenyl beta-D-cellobioside as the substrate. Mutation D124E increased the Km and decreased the Vmax. 5-fold each, while D124N reduced the Km 4.5-fold and the Vmax. 75-fold. The mutations had no effect on the cleavage mode of xylopentaose.
基于F家族已知木聚糖酶序列之间的相似性，对来自淡紫链霉菌的木聚糖酶A的三个不变酸性残基进行了研究。在对木聚糖酶A基因进行工程改造以使其能够表达后，在大肠杆菌中进行了定点诱变实验。用等排体形式（谷氨酰胺）取代Glu-128或Glu-236完全消除了木聚糖酶对木聚糖和对硝基苯基β-D-纤维二糖苷的酶活性，这表明这两种底物在同一位点被水解。这两个氨基酸可能代表催化残基。免疫学研究表明这两个突变体保留了相同的表位，这表明活性缺乏是突变的结果而非蛋白质错误折叠的结果。D124E突变不影响以木聚糖为底物时的动力学参数，但与野生型酶相比，D124N使Km降低了16倍，Vmax降低了14倍。当以对硝基苯基β-D-纤维二糖苷为底物时，这些突变的影响更为明显。D124E突变使Km增加，Vmax降低，两者均为5倍，而D124N使Km降低4.5倍，Vmax降低75倍。这些突变对木五糖的切割模式没有影响。
