SUMOylation plays important roles in many key physiological and pathological processes. The SUMOylation cascade involves a heterodimer of activating enzyme, E1 (Aos1/Uba2); a conjugating enzyme, E2 (Ubc9); and many ligase enzymes, E3. Focusing on the activation step of the SUMOylation process, we examined the interaction of E1 with its substrates. Previous studies reported the K-m of E1 enzymes in ubiquitin and other ubiquitin-like pathways, but the K-m of the SUMO paralogs (SUMO2 and SUMO3) is unknown. Here, by using quantitative FRET to measure the SUMO E1 enzyme kinetics of SUMO1, 2, and 3 and ATP under steady state conditions, we found that the enzyme kinetics from the quantitative FRET method are comparable to those from conventional radioactive assays. Additionally, the kinetic constants, K-m, of SUMO2 (3.418 +/- 0.9131M) and SUMO3 (2.764 +/- 0.75M) [FW1] are approximately four to five times higher than that of SUMO1 K-m (0.7458 +/- 0.1105M). These results demonstrate the advantages of FRET technology for determining K-m, including the ability to monitor reaction progress in real-time with high-throughput and high-sensitivity in an environmentally friendly manner. The processes discussed here extend the utility of quantitative FRET in characterizing protein-protein interactions and enzyme kinetics. Biotechnol. Bioeng. 2015;112: 652-658. (c) 2014 Wiley Periodicals, Inc.