title: Biochemical characterization of a multidomain deubiquitinating enzyme Ubp15 and the regulatory role of its terminal domains.
abstract: Deubiquitinating enzymes (DUBs) have emerged as essential players in a myriad of cellular processes, yet the regulation of DUB function remains largely unknown. While some DUBs rely on the formation of complex for regulation of enzymatic activity, many DUBs utilize interdomain interactions to regulate catalysis. Here we report the biochemical characterization of a multidomain deubiquitinating enzyme, Ubp15, from Saccharomyces cerevisiae. Steady-state kinetic investigation showed that Ubp15 is a highly active DUB. We identified active-site residues that are required for catalysis. We have also identified key residues on Ubp15 required for ubiquitin binding and catalysis. We further demonstrated that Ubp15's enzymatic activity is regulated by the N- and C-terminal domains that flank the catalytic core domain. Moreover, we demonstrated that Ubp15 physically interacts with a WD40 repeat-containing protein, Cdh1, by copurification experiments. Interestingly, unlike other DUBs that specifically interact with WD40 repeat-containing proteins, Cdh1 does not function in stimulating Ubp15's activity. The possible cellular function of Ubp15 in cell cycle regulation is discussed in view of the specific interaction between Ubp15 and Cdh1, an activator of the anaphase-promoting complex/cyclosome (APC/C).
authors.affiliation: Department of Chemistry and Biochemistry, University of Delaware, 214A Drake Hall, Newark, Delaware 19716, USA.
authors.forename: William P
authors.initials: WP
authors.lastname: Bozza
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Zhuang
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D064200:Cdh1 Proteins; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: C577583:CDH1 protein, S cerevisiae; D064200:Cdh1 Proteins; D050505:Mutant Proteins; D029701:Saccharomyces cerevisiae Proteins; D010450:Endopeptidases; C561120:Ubp15 protein, S cerevisiae