Summers Matthew K, Jackson Peter K
Department of Cellular Regulation, Genentech Inc., South San Francisco, CA, USA.
Methods Mol Biol. 2009;545:313-30. doi: 10.1007/978-1-60327-993-2_20.
The Anaphase Promoting Complex (APC) ubiquitin ligase is critical for multiple processes including cell cycle, development, meiosis, and senescence. The importance of regulation of the APC by substrate competitive (pseudosubstrate) inhibitors, such as Emi1 and BubR1, has recently been demonstrated. Substrate competitive inhibitors typically bind to enzymes via the same site as substrates, but by having any combination of increased enzyme affinity and low turnover numbers, are able to "clog" the ability of the enzyme to bind and turnover substrates. For the APC, these pseudosubstrates can both position and block the APC and have been well validated as critical regulators for the APC enzymes.We have found that the substrate competitive mechanism of inhibition is sensitive to the E2 activity driving APC catalyzed ubiquitination events. This chapter provides detailed protocols for multiple in vitro ubiquitination assays of increasing complexity and the use of pseudosubstrate inhibitors in these assays. These assays are instrumental in examining the use of E2 enzymes by the APC and the intimate relationship this has with pseudosubstrate inhibition.
后期促进复合物（APC）泛素连接酶对包括细胞周期、发育、减数分裂和衰老在内的多个过程至关重要。最近已证明底物竞争性（假底物）抑制剂（如Emi1和BubR1）对APC调节的重要性。底物竞争性抑制剂通常通过与底物相同的位点与酶结合，但通过具有增加的酶亲和力和低周转数的任何组合，能够“阻塞”酶结合和转化底物的能力。对于APC，这些假底物既可以定位并阻断APC，并且已被充分验证为APC酶的关键调节剂。我们发现抑制的底物竞争机制对驱动APC催化泛素化事件的E2活性敏感。本章提供了用于多种复杂性不断增加的体外泛素化测定以及在这些测定中使用假底物抑制剂的详细方案。这些测定有助于研究APC对E2酶的使用以及这与假底物抑制的密切关系。
