FUNCTIONAL ANALYSIS OF THE CATALYTIC DOMAIN OF BETA1-4GALACTOSYLTRANSFERASE

对于高尔基体催化结构域的结构分析 糖基转移酶，特别是β-1，4-半乳糖基转移酶 （β 1，4-GT），将半乳糖从UDP-半乳糖转移到N-半乳糖。 乙酰葡糖胺（NAG），游离的或与寡糖结合的， 酶的N-和C-末端缺失和点突变体的数量 在E.以确定结合区域的 与受体或供体底物相互作用的酶。 的 在残基1-129之间的酶的N-末端截短形式， 在对NAG或线性的表观Km中未显示任何差异 寡糖受体例如，对于壳二糖和壳三糖，或对于 核苷酸供体UDP-半乳糖。 Cys 134缺失或突变 导致酶活性丧失，但不影响 蛋白质与NAG-或UDP-琼脂糖的结合特性。 从 这些柱中的蛋白质可用15 mMNAG和50 mMEDTA洗脱， 如酶活性蛋白GT-129，其含有残基 130-402牛β-1，4 GT。 此外，N-末端片段，GT- 129 NAG含有β-1，4 GT的130-257位残基， ，并用15 mM NAG和50 mM EDTA从NAG-琼脂糖中洗脱 与酶活性GT-129一样有效。 不像 C-末端片段GT-257 UDP，含有残基258-402，N-末端片段GT-257 UDP， 末端片段GT-129 NAG，其含有的残基130-257 β-1，4GT与UDP-柱的结合效率较低， 仅用15 mM NAG从柱上洗脱。 另一方面，C- 末端片段GT-257 UDP与NAG-和UDP-均紧密结合， 琼脂糖柱。 一小部分，即5-10%的结合蛋白，可以被 从UDP-琼脂糖柱上用50 mM EDTA单独洗脱。结果 显示β-氨基端和C端片段的结合行为， 1，4GT对NAG-和UDP-琼脂糖柱的作用不同，前者 优先结合NAG-柱，而后者结合UDP- 琼脂糖柱通过Mn+2。

For the structural analysis of the catalytic domain of Golgi glycosyltransferases, in particular beta-1,4-galactosyltransferase (beta1,4-GT) that transfers galactose from UDP-galactose to N- acetylglucosamine (NAG), either free or bound to an oligosaccharide, a number of N- and C-terminal deletion and point mutants of the enzyme were expressed in E. coli to determine the binding regions of the enzyme that interact with the acceptor or donor substrates. The N-terminal truncated forms of the enzyme between the residues 1-129, do not show any difference in the apparent Km's towards NAG or linear oligosaccharide acceptors e.g., for chitobiose and chitotriose, or for the nucleotide donor UDP-galactose. Deletion or mutation of Cys 134 results in the loss of enzymatic activity, but does not affect the binding properties of the protein either to NAG- or UDP-agarose. From these columns the protein can be eluted with 15 mM NAG and 50 mM EDTA, like the enzymatically active protein, GT-129, that contains residues 130-402 of bovine beta-1,4GT. Also the N-terminus fragment, GT- 129NAG, that contains the residues 130-257 of the beta-1,4GT, binds to, and elutes with 15 mM NAG and 50 mM EDTA from the NAG-agarose column as efficiently as the enzymatically active GT-129. Unlike the C-terminus fragment GT-257UDP, containing residues 258-402, the N- terminus fragment, GT-129NAG, that contains the residues 130-257 of the beta-1,4GT, binds less efficiently to UDP-columns and can be eluted from the column with only 15 mM NAG. On the otherhand, the C- terminus fragment, GT-257UDP, binds tightly to both NAG- and UDP- agarose columns. A small fraction, 5-10% of the bound protein, can be eluted from the UDP-agarose column with 50 mM EDTA alone. The results show that the binding behavior of N- and C-terminal fragments of beta- 1,4GT towards the NAG- and UDP-agarose columns differ, the former binding preferentially to NAG-columns, while the latter binds to UDP- agarose columns via Mn+2.