| Assay Method Information | |
| | Biochemical Assay |
| Description: | In brief, this Rpn11 bioassay employs a fluorescent polarization readout based on the ability of the 26S proteasome to cleave the protein substrate including four tandem ubiquitin proteins fused to a peptide having a unique cysteine labeled with a fluorophore. Cleavage of this substrate by Rpn11 at the junction between the fourth ubiquitin and the peptide, releases the low molecular weight fluorescent peptide. Accordingly, inhibition of fluorescence correlates with inhibition of Rpn11. Inhibition is reported as the half maximal inhibitory concentration (IC50) for the candidate compound.The catalytic JAB1/MPN/Mov34 metalloenzyme (JAMM) motif of Rpn11 is found in 7 different human proteins including the Csn5 subunit of the COP9 signalosome, AMSH, AMSH-LP, the BRCC36 subunit of BRISC, MPND, and MYSM1. All of these enzymes cleave the isopeptide linkage that joins ubiquitin (or the ubiquitin-like protein Nedd8 in the case of Csn5) to a second molecule of ubiquitin or to a substrate. The conserved JAMM domain has the consensus sequence EXnHS/THX7SXXD, in which the histidine (His) and aspartic acid (Asp) residues bind the Zn2+ ion and the fourth coordination site is occupied by a water molecule that is engaged in hydrogen bonding with a conserved glutamic acid (Glu). The Zn2+ acts as a Lewis acid and increases the nucleophilic character of the bound water enough to allow hydrolytic cleavage of the isopeptide bond. |
| Affinity data for this assay | |
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