FUNCTIONAL ANALYSIS OF THE CATALYTIC DOMAIN OF BETA-1,4GALACTOSYLTRANSFERASE

我们继续进行结构分析工作， 在糖蛋白生物合成中起作用的糖基转移酶 和糖脂。 β-1，4-半乳糖基转移酶（β-1，4GT）是 位于高尔基体，在锰的存在下， 催化半乳糖从UDP-半乳糖转移到 糖蛋白的非还原性末端N-乙酰葡糖胺残基， 糖脂 根据动力学数据，已经表明， 需要金属离子，一个在催化过程中，另一个在催化过程中。 对蛋白质的结构完整性至关重要。 上一 今年，我们分别表达了N-和C-末端片段的 催化结构域，残基130-257和258-402，分别在 E.coli. 通过对复性片段的结合分析，我们表明， 糖受体的主要结合区域位于N-末端 部分，而核苷酸糖供体结合定位于 催化结构域的C-末端一半。 之间的二硫键 Cys 134和Cys 247在酶活性过程中是必需的， 对于与底物结合是必需的。 （Boeggeman等人， Glycoconjugate J 1995：12;865-78）。 Taq聚合酶的晶体结构分析揭示了保守的 二价锰结合所必需的序列基序， 催化作用 两个这样的位点位于N-（DYD和DVD）内， α 1，4GT的催化结构域的C-末端（EDDD）一半， 分别 我们采用定点突变技术分析了Mn ~（2+） β-1，4GT结合区。 我们的研究结果表明， 在242、244、252和254位， N-末端片段，消除β-1，4 GT的酶活性。 E317和位置318、319和320处的Asp残基的突变，也 破坏了酶的活性。 动力学分析表明， 突变体对UDP-半乳糖的表观Km增加50倍 D320 N与D244 N和野生型β-1，4GT相比。 这些结果 证明位于C-末端的D320突变 部分，仅影响与UDP-半乳糖底物的结合， N-乙酰葡萄糖胺，而作为D244的突变，位于 消除酶活性的N-末端部分不具有 对UDP-半乳糖或 N-乙酰葡糖胺

We have continued our work on the structural analyses of glycosyltransferases that function in the biosynthesis of glycoproteins and glycolipids. The beta-1,4-galactosyltransferase (beta-1,4GT) is localized in the trans Golgi, where in the presence of manganese, it catalyzes the transfer of galactose from UDP-galactose to the non-reducing terminal N-acetylglucosamine residue of glycoproteins and glycolipids. From the kinetic data it has been suggested that two metal ions are required, one during catalysis and the other being essential for the structural integrity of the protein. In the previous year we have separately expressed the N-and C-terminal fragments of the catalytic domain, residues 130-257, and 258-402, respectively, in E.coli. By binding analyses of the renatured fragments we showed that the major binding region for the sugar acceptor lies in the N-terminal portion while the nucleotide sugar donor binding is localized to the C-terminal half of the catalytic domain. The disulfide bond between Cys134 and Cys247 is required during enzymatic activity and is not essential for binding to the substrates. (Boeggeman et al., Glycoconjugate J 1995:12;865-78). Crystal structure analysis of the Taq polymerase has revealed conserved sequence motifs which are essential for divalent manganese binding and catalysis. Two such sites are localized within the N-(DYD and DVD) and C-terminal (EDDD) halves of the catalytic domain of a1,4GT, respectively. We have used site directed mutagenesis to analyze Mn2+ binding regions of beta-1,4GT. Our results demonstrate that mutation of Asp residue at positions 242, 244, 252 and 254, within the N-terminal fragment, abrogate the enzymatic activity of beta-1,4GT. Mutation of E317 and Asp residue at positions 318, 319 and 320, also destroyed the enzymatic activity. Furthermore, kinetic analyses show 50 fold increase in the apparent Km for UDP-galactose of the mutant D320N compared to D244N and the wild type beta-1,4GT. These results demonstrate that mutation of D320, located within the C-terminal portion, influences only the binding to UDP-galactose substrate and not to N-acetylglucosamine, while as mutation of D244, located within the N-terminal portion, that abrogates the enzymatic activity, has no influence on the binding of either UDP-galactose or N-acetylglucosamine.