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

β-1,4半乳糖基转移酶催化域的功能分析

• 批准号: 2463740
• 负责人: P K QASBA
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2463740
• 关键词: Golgi apparatus; active sites; disulfide bond; enzyme activity; enzyme substrate; galactosyltransferases; manganese; protein structure function; site directed mutagenesis

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.

我们继续我们在结构分析方面的工作 糖基转移酶在糖蛋白生物合成中的作用 和糖脂。β-1，4-半乳糖基转移酶(β-1，4GT)是 定位于外高尔基山脉，在那里有锰存在，它 催化半乳糖从UDP-半乳糖转移到 糖蛋白的非还原末端N-乙酰氨基葡萄糖残基和 糖脂。从动力学数据中可以看出，有两个 金属离子是必需的，一个是在催化过程中，另一个是 对于蛋白质的结构完整性是必不可少的。在上一次 去年，我们已经分别表达了N端和C端的片段 催化结构域，残基分别为130-257和258-402，在 大肠埃希菌。通过对复性片段的结合分析，我们发现 糖受体的主要结合区在N-末端 部分，而核苷酸糖供体结合定位于 催化结构域的C-末端的一半。分子间的二硫键 Cys134和Cys247是酶活性所必需的，而不是 与底物结合所必需的。(Boeggeman等人， 糖共轭J 1995：12；865-78)。 Taq聚合酶的晶体结构分析表明它是保守的 二价锰结合所必需的序列基序和 催化作用。有两个这样的站点位于N-(DYD和DVD)和 A1，4GT催化域的C末端(EDDD)半部分， 分别进行了分析。我们利用定点突变技术分析了Mn2+ β-1，4GT的结合区。我们的结果表明，突变 天冬氨酸残基在第242、244、252和254位 N-末端片段，消除了β-1，4GT的酶活性。 E317和Asp残基318、319和320位的突变 破坏了酶的活性。此外，动力学分析表明， 突变株UDP-半乳糖的表观Km增加50倍 D320N与D244N和野生型β-1，4GT的比较。这些结果 证明了位于C-末端的D320突变 部分，只影响与UDP-半乳糖底物的结合，而不影响 为N-乙酰氨基葡萄糖，而作为D244的突变，位于 取消酶活性的N-末端部分没有 对UDP-半乳糖或UDP-Gal结合的影响 N-乙酰氨基葡萄糖。