Deubiquitinating enzymes (DUBs) are important regulators that remove ubiquitin, a protein that attaches to other proteins and helps in controlling various cellular functions such as endocytosis, DNA repair, and protein degradation. SST2, which belongs to the JAMM metalloprotease family of DUBs, is an AMSH orthologue in fission yeast and is involved in endosomal trafficking and lysosomal degradation. In this project, we are interested in understanding the role of the loop near the active site that opens and closes during its catalytic cycle. To understand its functions, we deleted the active site loop residues and studied its effect on the enzymatic activity. The mutant showed defect in both Kcat and Km in compared to wild type Sst2. The reduction in Kcat indicates the loss of interaction with the isopeptide bond of dibuiquitin substrate whereas; increase in Km may be due to the loss of interaction with the proximal ubiquitin. Circular Dichroism spectroscopy will be performed to study if there is any structural difference that results in the loss of activity. The results from this study will help us understand that active site loop are important for both catalysis and substrate recognition.

Function of an active site loop in a metallo deubiquitinase, SST2

251st National Meeting (2016)

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