FUNCTIONAL ANALYSIS OF THE CATALYTIC DOMAIN OF BETA1-4GALACTOSYLTRANSFERASE

牛β 1，4半乳糖基转移酶（beta1，4GT）是一个长402个残基的酶 蛋白质，其具有与其他蛋白质类似的一般拓扑结构， 糖基转移酶，由一个短的氨基末端胞质 尾、膜信号锚结构域、茎区和羧基- 末端催化结构域 这种酶将半乳糖从UDP- 半乳糖转化为N-乙酰葡糖胺（NAGlc），无论是游离的还是结合到 寡糖，以产生具有β 1 -4键的N-乙酰基乳糖胺。 为了分析催化结构域，我们制备了氨基- 将其末端缺失的蛋白克隆到pGEX-2 T载体中进行表达 在大肠coli中表达谷胱甘肽-S-转移酶（GST）融合蛋白。 重组蛋白定位于包涵体中， 溶解于5 M盐酸胍中。 的复性和再生 酶活性，从溶解的蛋白质是严格依赖于 存在“氧化改组”试剂，8：1 mM 还原型：氧化型谷胱甘肽。 复性/再氧化融合蛋白， 在谷胱甘肽亲和柱上纯化，在β 1，4 GT和 乳糖合成酶（LS）测定。 凝血酶裂解融合后 蛋白和随后的GST结构域的去除，重组β 1，4GT 具有牛乳GT比活性的50-85%。 NAGlc和UDP-半乳糖的含量与牛乳β 1，4GT相似。 缺失分析表明，β 1，4GT和LS活性保持完整 即使在缺少前129个残基的情况下， 当缺失延伸至残基142时。 Cys定点突变 134转丙氨酸或丝氨酸导致β 1，4 GT和LS的丢失 酶活性 与之前报道的结果（Aoki D， Appert HE，约翰逊D，王55，福田MN（1990）EMBO J. 9，3171-3178），我们的 结果表明，大肠杆菌中产生的β 1，4 GT以包涵体形式存在 作为一种非活性蛋白质。需要“氧化改组”试剂， 涉及Cys 134的二硫键的形成，这对于 蛋白质的正确折叠和酶的再生 活动

Bovine beta1,4galactosyltransferase (beta1,4GT) is a 402 residue long protein, which has a general topology similar to other glycosyltransferases that consists of a short amino-terminal cytoplasmic tail, a membrane signal anchor domain, a stem region and a carboxyl- terminal catalytic domain. The enzyme transfers galactose from UDP- galactose to N-acetylglucosamine (NAGlc), either free or bound to an oligosaccharide, to produce N-acetyllactosamine with a beta1-4 linkage. To analyze the catalytic domain we prepared cDNA constructs of the amino- terminal deleted forms of the protein in pGEX-2T vector and expressed them in E. coli as glutathione-S-transferase (GST) fusion proteins. Recombinant proteins were localized in inclusion bodies which were solubilized in 5 M guanidine-HCl. Renaturation and regeneration of the enzyme activity, from the solubilized protein was strictly dependent on the presence of an "oxido-shuffling" reagent, a mixture of 8:1 mM reduced:oxidized glutathione. Renatured/reoxidized fusion proteins, purified on glutathione-affinity columns, were active in beta1,4GT and lactose synthetase (LS) assays. After thrombin cleavage of the fusion protein and subsequent removal of GST domain, the recombinant beta1,4GT has 50-85% of the specific activity of bovine milk GT. The apparent K-m's for NAGlc and UDP-galactose were similar to that of bovine milk beta1,4GT. Deletion analysis show that both beta1,4GT and LS activities remain intact even in the absence of the first 129 residues, but the activities are lost when deletions extend up to residue 142. Site directed mutagenesis of Cys 134 to either Ala or Ser resulted in the loss of both beta1,4GT and LS enzyme activities. In contrast to the previously reported result (Aoki D, Appert HE, Johnson D, Wang 55, Fukuda MN (1990) EMBO J. 9, 3171-3178), our results show that beta1,4GT produced in E.coli is present in inclusion bodies as an inactive protein. "Oxido-shuffling" reagents are required for the formation of a disulfide bond involving Cys 134, which is crucial for proper folding of the protein and for the regeneration of the enzyme activity.