Core domain mutation (S86Y) selectively inactivates polyubiquitin chain synthesis catalyzed by E2-25K
Core domain mutation (S86Y) selectively inactivates polyubiquitin chain synthesis catalyzed by E2-25K
复制标题
DOI：
10.1021/bi9800911
复制
发表时间：
1998-07-07
期刊：
影响因子：
2.9
通讯作者：
Pickart, CM
中科院分区：
文献类型：
--
作者：
Mastrandrea, LD;Kasperek, EM;Pickart, CM
哺乳动物泛素偶联酶E2-25K催化合成通过K48-G76异肽键连接的多泛素链。截断和嵌合形式E2- 25k的性质表明，该E2的多泛素链合成活性取决于其保守的150个残基核心结构域和独特的50个残基尾结构域之间的特定相互作用[Haldeman， M. T., Xia， G., Kasperek， E. M., and Pickart， C. M. (1997) Biochemistry 36, 10526-10537]。在本研究中，我们通过显示核心结构域（S86Y）的点突变模拟了删除整个尾部结构域的效果，为该模型提供了强有力的支持：形成类似于泛素硫醇酯的E2的能力完好无损，而偶联活性受到严重抑制（大于或等于k(cat)/ k -m降低100倍）。携带S86Y突变的E2-25K酶的特性表明，这种突变加强了核心和尾部结构域之间的相互作用：S86Y- 25k的游离形式和泛素结合形式都完全抵抗尾结构域K164位点的色氨酸切割，而野生型酶在该位点快速切割。S86Y-26K的其他特性表明，该突变型酶的活性位点比野生型酶的活性位点更闭塞。(1)游离S86Y-25K被碘乙酰胺烷基化的速度比野生型慢2倍。(2)在E2类似于泛素硫醇酯形成的实验中，S86Y-25K对E1的亲和力降低了4倍。(3) S86Y-25K的泛素硫醇酯加合物与二硫苏糖醇的（非催化）反应速度比野生型硫醇酯加合物慢3倍。一种适应这些发现的模型假设，由S86Y突变诱导的核心和尾部结构域之间的增强相互作用导致活性位点的位阻，从而阻止进入的泛素受体进入硫醇酯键。与该模型一致，S86Y突变抑制泛素向大分子受体（泛素和聚赖氨酸）的转移比向小分子受体（游离赖氨酸和短肽）的转移更强烈。这些结果表明，E2活性位点附近的独特残基可能通过介导分子内相互作用来影响特定功能。
The mammalian ubiquitin conjugating enzyme known as E2-25K catalyzes the synthesis of polyubiquitin chains linked exclusively through K48-G76 isopeptide bonds. The properties of truncated and chimeric forms of E2-25K suggest that the polyubiquitin chain synthesis activity of this E2 depends on specific interactions between its conserved 150-residue core domain and its unique 50-residue tail domain [Haldeman, M. T., Xia, G., Kasperek, E. M., and Pickart, C. M. (1997) Biochemistry 36, 10526-10537]. In the present study, we provide strong support for this model by showing that a point mutation in the core domain (S86Y) mimics the effect of deleting the entire tail domain: the ability to form an E2 similar to ubiquitin thiol ester is intact, while conjugation activity is severely inhibited (greater than or equal to 100-fold reduction in k(cat)/K-m). The properties of E2-25K enzymes carrying the S86Y mutation indicate that this mutation strengthens the interaction between the core and tail domains: both free and ubiquitin-bound forms of S86Y-25K are completely resistant to tryptic cleavage at K164 in the tail domain, whereas wild-type enzyme is rapidly cleaved at this site. Other properties of S86Y-26K suggest that the active site of this mutant enzyme is more occluded than the active site of the wild-type enzyme. (1) Free S86Y-25K is alkylated by iodoacetamide 2-fold more slowly than the wild-type enzyme. (2) In assays of E2 similar to ubiquitin thiol ester formation, S86Y-25K shows a 4-fold reduced affinity for E1. (3) The ubiquitin thiol ester adduct of S86Y-25K undergoes (uncatalyzed) reaction with dithiothreitol 3-fold mon slowly than the wild-type thiol ester adduct. One model to accommodate these findings postulates that an enhanced interaction between the core and tail domains, induced by the S86Y mutation, causes a steric blockade at the active site which prevents access of the incoming ubiquitin acceptor to the thiol ester bond. Consistent with this model, the S86Y mutation inhibits ubiquitin transfer to macromolecular accepters (ubiquitin and polylysine) more strongly than transfer to small-molecule accepters (free lysine and short peptides). These results suggest that unique residues proximal to E2 active sites may influence specific function by mediating intramolecular interactions.