Tamada Y, Swanson B A, Arabshahi A, Frey P A
Institute for Enzyme Research, Graduate School, University of Wisconsin-Madison 53705.
Bioconjug Chem. 1994 Nov-Dec;5(6):660-5. doi: 10.1021/bc00030a023.
A fusion enzyme consisting of UDP-galactose 4-epimerase and galactose-1-P uridylyltransferase with an intervening Ala3 linker was constructed by in-frame fusion of E. coli gene galT to the 3'-terminus of the E. coli gene galE that had been extended with the coding sequence for three alanine residues, all contained within a high-expression plasmid. The fusion enzyme was expressed in E. coli and purified 24-fold to about 98% homogeneity by chromatography on hydroxylapatite and Q-Sepharose. On the basis of the comparison of the elution profile for enzyme activities upon gel permeation chromatography (Sephacryl S-400) with the molecular weight of 80,000 determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, the fusion enzyme appears to exist in monomeric, dimeric, and tetrameric forms, all of which exhibit both enzymatic activities. The Km values of the fusion enzyme for substrates were similar to those for the corresponding native enzymes, except for UDP-glucose, but the kcat values were smaller than those for the native enzymes. The fusion enzyme shows kinetic advantages in that the initial velocity to produce glucose-1-P from UDP-galactose and galactose-1-P is about 20% faster than that for a mixture of equal activities of the separate enzymes.
一种由UDP-半乳糖4-差向异构酶和1-磷酸半乳糖尿苷酰转移酶组成的融合酶，中间有Ala3接头，通过将大肠杆菌基因galT读框内融合到已用三个丙氨酸残基编码序列延伸的大肠杆菌基因galE的3'末端构建而成，所有这些都包含在一个高表达质粒中。该融合酶在大肠杆菌中表达，并通过羟基磷灰石和Q-琼脂糖柱层析纯化24倍，纯度约为98%。根据凝胶渗透色谱法（Sephacryl S-400）上酶活性洗脱图谱与十二烷基硫酸钠聚丙烯酰胺凝胶电泳测定的80,000分子量的比较，融合酶似乎以单体、二聚体和四聚体形式存在，所有这些形式都具有两种酶活性。融合酶对底物的Km值与相应天然酶相似，但UDP-葡萄糖除外，但其kcat值小于天然酶。融合酶具有动力学优势，即从UDP-半乳糖和1-磷酸半乳糖产生1-磷酸葡萄糖的初始速度比单独酶等活性混合物快约20%。
