Mikkelsen R, Binderup K, Preiss J
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Arch Biochem Biophys. 2001 Jan 15;385(2):372-7. doi: 10.1006/abbi.2000.2164.
Branching enzyme belongs to the alpha-amylase family, which includes enzymes that catalyze hydrolysis or transglycosylation at alpha-(1,4)- or alpha-(1,6)-glucosidic linkages. In the alpha-amylase family, four highly conserved regions are proposed to make up the active site. From amino acid sequence analysis a tyrosine residue is completely conserved in the alpha-amylase family. In Escherichia coli branching enzyme, this residue (Y300) is located prior to the conserved region 1. Site-directed mutagenesis of the Y300 residue in E. coli branching enzyme was used in order to study its possible function in branching enzymes. Replacement of Y300 with Ala, Asp, Leu, Ser, and Trp resulted in mutant enzymes with less than 1% of wild-type activity. A Y300F substitution retained 25% of wild-type activity. Kinetic analysis of Y300F showed no effect on the Km value. The heat stability of Y300F was analyzed, and this was lowered significantly compared to that of the wild-type enzyme. Y300F also showed lower relative activity at elevated temperatures compared to wild-type. Thus, these results show that Tyr residue 300 in E. coli branching enzyme is important for activity and thermostability of the enzyme.
分支酶属于α-淀粉酶家族，该家族包括催化α-(1,4)-或α-(1,6)-糖苷键水解或转糖基化反应的酶。在α-淀粉酶家族中，四个高度保守的区域构成了活性位点。通过氨基酸序列分析发现，酪氨酸残基在α-淀粉酶家族中完全保守。在大肠杆菌分支酶中，该残基（Y300）位于保守区域1之前。为了研究其在分支酶中的可能功能，对大肠杆菌分支酶中的Y300残基进行了定点诱变。用丙氨酸、天冬氨酸、亮氨酸、丝氨酸和色氨酸取代Y300，得到的突变酶活性不到野生型的1%。Y300F取代保留了25%的野生型活性。对Y300F的动力学分析表明，其Km值没有变化。对Y300F的热稳定性进行了分析，与野生型酶相比，其热稳定性显著降低。与野生型相比，Y300F在高温下也表现出较低的相对活性。因此，这些结果表明，大肠杆菌分支酶中的酪氨酸残基300对该酶的活性和热稳定性很重要。
