Because the E enzyme directly recognizes

Because the E2 enzyme directly recognizes a SCM, it can modify substrates in vitro at high enzyme concentrations as we illustrate in Fig. 1A. Hence, it is important to use a low E2 concentration in E3-dependent reactions that either show no or only marginal substrate modification by the E2 alone. At high enzyme concentrations, the mammalian E2 itself gets sumoylated at Lys 14 (S*E2). We have shown that this particular modification stabilizes the E2 interaction with GST-Sp100 due to a SIM in the substrate that is in close proximity to the SCM. The E2*S adduct therefore also enhances GST-Sp100 modification to a degree comparable to some E3 ligases (Knipscheer et al., 2008) (Fig. 1A). However, E3 ligases are usually more potent and promote higher sumoylation rates when at low (substoichiometric) levels; this can be seen both by varying E3 concentrations (Fig. 1B) and by following sumoylation over time (Fig. 1C).
E3-mediated E2 discharge E3 ligases simultaneously interact with the substrate and the SUMOD charged E2 enzyme to catalyze the discharge of the thioester-bound SUMOD from the E2 to the substrate. E3 interaction with SUMOD via a SIM results in a closed conformation which is highly reactive and leads to rapid discharge of the thioester bond as it was shown for all three gdc com receptor of bona fide SUMO E3 ligases (Cappadocia et al., 2015; Eisenhardt et al., 2015; Reverter & Lima, 2005; Streich & Lima, 2016). By using E1 and E2 enzymes together with ATP, Mg, and SUMO, a SUMOD~E2 thioester is formed. To monitor the discharge of SUMOD from the E2, ATP needs to be hydrolyzed by apyrase (an ATP diphosphohydrolase) to prevent recharging of the E2. As this assay allows only a single round of E2 discharge (called a “single-turnover” assay), it requires much higher enzyme concentrations compared to the “multiturnover” reactions described in Fig. 1 that cycle through multiple rounds of charging and discharging. The major challenge of single-turnover reactions is the ability of the E2 to also be discharged in the absence of an E3, and it discharges within minutes at 30°C. This is likely due to the high E2 enzyme concentration that allows E3-independent modification of the E1, the E2, SUMO, and the substrate. Hence, E3-dependent discharge has to be executed at high E3 concentrations and in a short time frame to visualize SUMOD~E2 discharge and substrate modification (Fig. 2).
E3–E2 backside interaction The E2 possesses an important regulatory interface which is termed its backside as it is opposite to the catalytic cleft that bears the active-site cysteine forming the thioester with SUMOD. This backside site interacts noncovalently with a scaffold SUMOB and was originally shown to be important for E2-mediated SUMO chain formation in vitro (Capili & Lima, 2007; Knipscheer, van Dijk, Olsen, Mann, & Sixma, 2007). Moreover, it partially overlaps with the E2–E1 interface (Duda et al., 2007) and is required for direct or indirect E2–E3 interactions. SUMOB is essential for the E3 activity of ZNF451 family members (Cappadocia et al., 2015; Eisenhardt et al., 2015) and strongly enhances the activity of Siz/Pias family members (Mascle et al., 2013; Streich & Lima, 2016), whereas RanBP2 directly interacts with the backside of the E2 independent of a SUMO (Pichler et al., 2004; Reverter & Lima, 2005).