Mastrandrea L D, Kasperek E M, Niles E G, Pickart C M
Department of Biochemistry, State University of New York, Buffalo 14214, USA.
Biochemistry. 1998 Jul 7;37(27):9784-92. doi: 10.1021/bi9800911.
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 approximately ubiquitin thiol ester is intact, while conjugation activity is severely inhibited (>/=100-fold reduction in kcat/Km). 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 approximately 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 more 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 acceptors (ubiquitin and polylysine) more strongly than transfer to small-molecule acceptors (free lysine and short peptides). These results suggest that unique residues proximal to E2 active sites may influence specific function by mediating intramolecular interactions.
被称为E2-25K的哺乳动物泛素缀合酶催化仅通过K48-G76异肽键连接的多聚泛素链的合成。E2-25K截短形式和嵌合形式的特性表明，该E2的多聚泛素链合成活性取决于其保守的150个残基核心结构域与其独特的50个残基尾部结构域之间的特定相互作用[哈德曼，M.T.，夏，G.，卡斯佩雷克，E.M.，和皮卡德，C.M.（1997年）《生物化学》36，10526 - 10537]。在本研究中，我们通过表明核心结构域中的一个点突变（S86Y）模拟了删除整个尾部结构域的效果，为该模型提供了有力支持：形成E2 - 泛素硫酯的能力完好无损，而缀合活性受到严重抑制（kcat/Km降低≥100倍）。携带S86Y突变的E2-25K酶的特性表明，该突变增强了核心结构域与尾部结构域之间的相互作用：S86Y - 25K的游离形式和与泛素结合的形式对尾部结构域中K164处的胰蛋白酶切割完全具有抗性，而野生型酶在此位点迅速被切割。S86Y - 26K的其他特性表明，该突变酶的活性位点比野生型酶的活性位点更封闭。（1）游离的S86Y - 25K被碘乙酰胺烷基化的速度比野生型酶慢2倍。（2）在E2 - 泛素硫酯形成的测定中，S86Y - 25K对E1的亲和力降低了4倍。（3）S86Y - 25K的泛素硫酯加合物与二硫苏糖醇的（非催化）反应速度比野生型硫酯加合物慢3倍。一个解释这些发现的模型假设，由S86Y突变诱导的核心结构域与尾部结构域之间增强的相互作用在活性位点处造成了空间位阻，阻止了进入的泛素受体接近硫酯键。与该模型一致，S86Y突变对泛素向大分子受体（泛素和多聚赖氨酸）的转移抑制作用比对小分子受体（游离赖氨酸和短肽）的转移更强。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。
