Isopeptidase Kinetics Determination by a Real Time and Sensitive qFRET Approach
| Autor: | Lei Xu, Yang Song, J. Jefferson P. Perry, Yali Shen, Yan Liu, Jiayu Liao |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Proteases
SENP1 medicine.medical_treatment SUMO protein Biochemistry Microbiology Sensitivity and Specificity Article enzyme kinetics Catalytic Domain Carbon-Nitrogen Lyases medicine Fluorescence Resonance Energy Transfer Humans Enzyme kinetics Molecular Biology Enzyme Assays chemistry.chemical_classification Protease Chemistry SENP Sumoylation QR1-502 isopeptidase Recombinant Proteins Isopeptidase activity quantitative FRET (qFRET) assay Cysteine Endopeptidases Kinetics Förster resonance energy transfer Enzyme Plasmids |
| Zdroj: | Biomolecules Volume 11 Issue 5 Biomolecules, Vol 11, Iss 673, p 673 (2021) |
| ISSN: | 2218-273X |
| Popis: | Isopeptidase activity of proteases plays critical roles in physiological and pathological processes in living organisms, such as protein stability in cancers and protein activity in infectious diseases. However, the kinetics of protease isopeptidase activity has not been explored before due to a lack of methodology. Here, we report the development of novel qFRET-based protease assay for characterizing the isopeptidase kinetics of SENP1. The reversible process of SUMOylation in vivo requires an enzymatic cascade that includes E1, E2, and E3 enzymes and Sentrin/SUMO-specific proteases (SENPs), which can act either as endopeptidases that process the pre-SUMO before its conjugation, or as isopeptidases to deconjugate SUMO from its target substrate. We first produced the isopeptidase substrate of CyPet-SUMO1/YPet-RanGAP1c by SUMOylation reaction in the presence of SUMO E1 and E2 enzymes. Then a qFRET analyses of real-time FRET signal reduction of the conjugated substrate of CyPet-SUMO1/YPet-RanGAP1c to free CyPet-SUMO1 and YPet-RanGAP1c by the SENP1 were able to obtain the kinetic parameters, Kcat, KM, and catalytic efficiency (Kcat/KM) of SENP1. This represents a pioneer effort in isopeptidase kinetics determination. Importantly, the general methodology of qFRET-based protease isopeptidase kinetic determination can also be applied to other proteases. |
| Databáze: | OpenAIRE |
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