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Determination of SUMO1 and ATP affinity for the SUMO E1by quantitative FRET technology
Author(s) -
Wiryawan Hilda,
Dan Kevin,
Etuale Myra,
Shen Yali,
Liao Jiayu
Publication year - 2015
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.25480
Subject(s) - sumo protein , förster resonance energy transfer , kinetics , enzyme , enzyme kinetics , dna ligase , biophysics , chemistry , biochemistry , single molecule fret , ubiquitin ligase , ubiquitin , biology , fluorescence , active site , gene , physics , quantum mechanics
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 ± 0.9131 μM) and SUMO3 (2.764 ± 0.75 μM) [FW1] are approximately four to five times higher than that of SUMO1 K m (0.7458 ± 0.1105 μM). 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. © 2014 Wiley Periodicals, Inc.