The intramembrane active site of GlpG, an E-coli rhomboid protease, is accessible to water and hydrolyses an extramembrane peptide bond of substrates
The intramembrane active site of GlpG, an E-coli rhomboid protease, is accessible to water and hydrolyses an extramembrane peptide bond of substrates
复制标题
DOI：
10.1111/j.1365-2958.2007.05679.x
复制
发表时间：
2007-04-01
影响因子：
3.6
通讯作者：
Akiyama, Yoshinori
中科院分区：
文献类型：
--
作者：
Maegawa, Saki;Koide, Kayo;Akiyama, Yoshinori
大肠杆菌 GlpG 是菱形蛋白酶的直系同源物，可催化特定膜蛋白的膜内蛋白水解。我们之前表明，它可以在体内和体外裂解 I 型模型膜蛋白 Bla-LY2-MBP，该蛋白在 LY2 的预测周质膜边界区域具有乳糖通透酶 (LY2) 的第二跨膜区域。在这里，我们通过检查用膜可渗透和不可渗透的烷基化试剂引入催化残基周围区域的Cys残基的可修饰性，研究了膜集成状态下GlpG活性位点区域的环境。结果表明酶活性位点完全向外部水相开放。 GlpG 还切割类似的融合蛋白 Bla-GknTM-MBP，其具有 Gurken (GknTM) 的跨膜区域，Gurken 是果蝇菱形的生理底物。 LY2和GknTM序列的切割位点区域中的工程Cys残基被膜不可渗透的烷基化试剂有效地修饰，表明这些区域暴露于周质。这些结果表明，GlpG 切割底物的膜外区域，这与目前流行的观点不同，即此类膜蛋白酶在横向进入酶活性位点后对膜嵌入的多肽片段起作用。
Escherichia coli GlpG is an orthologue of the rhomboid proteases that catalyse intramembrane proteolysis of specific membrane proteins. We previously showed that it can cleave a type I model membrane protein, Bla-LY2-MBP, having the second transmembrane region of lactose permease (LY2) in vivo and in vitro at the predicted periplasm-membrane boundary region of LY2. Here we investigated the environment of the active site regions of GlpG in the membrane-integrated state by examining the modifiability of Cys residues introduced into the regions around the catalytic residues with membrane-permeable and -impermeable alkylating reagents. The results indicate that the enzyme active site is fully open to the external aqueous phase. GlpG also cleaved a similar fusion protein, Bla-GknTM-MBP, having the transmembrane region of Gurken (GknTM), a physiological substrate of Drosophila rhomboids. Engineered Cys residues in the cleavage site regions of the LY2 and GknTM sequences were efficiently modified with a membrane-impermeable alkylating reagent, showing that these regions are exposed to the periplasm. These results suggest that GlpG cleaves an extramembrane region of substrates, unlike the currently prevailing view that this class of membrane proteases acts against a membrane-embedded polypeptide segment after its lateral entrance into the enzyme's active site.