Flores, Rosemary2017-06-272017-06-272016http://udspace.udel.edu/handle/19716/21508Deubiquitinating enzymes (DUBs) are imperative participants in multiple cellular processes, and as a consequence the control of DUB function has become the subject of great interest due to its possible implications in neurodegenerative diseases, anti-viral/bacterial responses, and potential anti-cancer targets. Recently a new DUB, USP30, was shown to be localized in the mitochondria and identified as having a role in the regulation of mitochondrial mitophagy, whose dysfunction is associated with the neurodegenerative Parkinson’s disease. The ubiquitin specific proteases (USPs), cysteine proteases, have a conserved catalytic core including cysteine, histidine and aspartate/asparagine. USP30 is very similar to other DUBs, except for the Asp/Asn conserved residue which is a serine. Our efforts were to understand how this unconventional catalytic residue in the catalytic core plays a role in the control of deubiquitination by first characterizing its steady state kinetics. A truncated USP30 was cloned into a pSUMO vector for expression in E.coli to obtain the wild-type (WT) turnover rate. Mutations in the catalytic triad were made to further understand the influence of the serine in the catalytic core. The WT kcat/KM was estimated to be 3.1 x 104 M-1s-1, while the C77A USP30 mutant had no detectable activity, as expected, and the S477A USP30 mutation had an estimated kcat/ KM of 2.2 x102 M-1s -1. Our results give insight to the importance of the serine and its possible role in regulating the rate of the USP30.Cysteine proteinases.Cysteine proteinases -- Purification.Enzymes.Catalysis.Serine.Chemical kinetics.Escherichia coli.Molecular cloning.Gene expression.Thesis993259643