FIGURE 5.
E1 mutants do not alter the adenylation and thioester formation activities. a, formation of the RanGAP1-SUMO conjugate catalyzed by the wild-type (WT) and mutant E1. Fifteen μm SUMO was incubated (15 min, 37 °C) with the ATP-regeneration system, 15 μm RanGAP1, 0.5 μm Ubc9, and 0.5 μm E1 or one of the E1 mutants at the indicated concentrations. The assays were resolved by SDS-PAGE and stained with SimpleBlue. Proteins of interest are marked with arrowheads. b and c, ATP:PPi exchange and ATP:AMP exchange rates of the wild-type and mutant E1 enzymes. The rates are normalized to the percentage of the E1 enzyme that is active, judged by the percentage of E1 that can form thioester conjugates with SUMO (see details in text). d, time-dependent formation of SUMO-E1 thioester conjugate (details in text).