Arutyunova E, Strisovsky K, Lemieux M J
Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton, AB, Canada.
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Methods Enzymol. 2017;584:395-437. doi: 10.1016/bs.mie.2016.11.002. Epub 2016 Dec 8.
Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of K and V to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein.
菱形蛋白酶是普遍存在的膜内丝氨酸蛋白酶，参与多种信号通路。这类迷人的蛋白酶含有一个埋藏在脂质环境中的活性位点。大肠杆菌菱形蛋白酶GlpG与各种抑制剂的高分辨率结构揭示了菱形蛋白酶介导的蛋白水解的催化机制；然而，尚缺乏定量表征。评估一种酶的催化参数对于理解其蛋白水解反应和调控机制的细节很重要。为了测定菱形蛋白酶活性，存在许多挑战，如脂质环境和缺乏已知底物。在这里，我们总结了过去十年中为研究菱形蛋白酶活性而开发的各种酶促测定方法。我们提供了基于凝胶迁移和荧光共振能量转移（FRET）测定的详细方案，以及使用去污剂溶解的菱形蛋白酶与细菌菱形蛋白酶唯一已知底物TatA和模型底物荧光标记酪蛋白计算米氏常数（K）和最大反应速率（V）以测量催化参数的方法。
