Ubiquitin-AMC showing 7-amino-4-methylcoumarin bound to the C-terminus of ubiquitin

Ubiquitin-AMC is a fluorogenic substrate for a wide range of deubiquitinating enzymes (DUBs), including ubiquitin C-terminal hydrolases (UCHs) and ubiquitin specific proteases (USPs). It is a particularly useful reagent for the study of deubiquitinating activity where detection sensitivity or continuous monitoring of activity is essential.[1]

Background

Ubiquitin-AMC is prepared by the C-terminal derivatization of ubiquitin with 7-amino-4-methylcoumarin and has been shown to be a useful and sensitive fluorogenic substrate for wide range of deubiquitinylating enzymes (DUBs), including ubiquitin C-terminal hydrolases (UCHs) and ubiquitin specific proteases (USPs).

Ubiquitin-AMC has been shown to be a sensitive substrate for UCH-L3 (Km = 0.039µM) and for Isopeptidase-T (Km = 0.17-1.4µM),[2] and is particularly useful for studying deubiquitinylating activity where detection sensitivity or continuous monitoring of activity is essential.[3]

Typical assay set-up: Assay substrate concentration: 0.01-1.0µM. Enzyme concentrations, UCH-L3: 10-100pM, isopeptidase-T: 10-100nM. Release of AMC fluorescence by DUB enzymes can be monitored using 380 nm excitation and 460 nm emission wavelengths.

Uses

  1. Substrate for deubiquitinylating enzyme activity assays.
  2. Identification/confirmation of enzyme deubiquitinylation activity.
  3. Investigation of deconjugating enzyme substrate specificity in comparison with alternative UBL-AMC substrates (e.g. NEDD8-AMC)

References

  1. ↑ BioMol Online Catalog
  2. ↑ Dang, L.C., Melandri, F.D. and Stein, R.L. Kinetic and mechanistic studies on the hydrolysis of ubiquitin C-terminal 7-amido-4-methylcoumarin by deubiquitinating enzymes. Biochemistry, 37, 1868-1879 (1998)
  3. ↑ Mason, D.E., Ek, J., Peters, E.C. and Harris J.L. Substrate profiling of deubiquitin hydrolases with a positional scanning library and mass spectrometry. Biochemistry, 43, 6535-44 (2004)
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