Study On The Activity And Kinetics Of Ubiquitin C-terminal Hydrolase

Ubiquitin C-terminal hydrolases (UCHs) belong to a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we describe a convenient method by the subustrates of Phe45Trp Ub-fusion proteins (Ub^F45W-Xaa) for characterizing substrate specificities of various UCHs. After removal of the His6-tagged proteins by Ni-NTA affinity, the enzymatic activities of the UCHs are quantitatively determined by fluorescence assay of the Ub^F45W product. The results suggest that three UCHs, UCH-L1, UCH-L3 and UCH37 have discrimination in their substrate specificities for the Ub extension proteins. This method may also be applicable to testing the substrate specificities of other DUBs.