title: Transient kinetic analysis of USP2-catalyzed deubiquitination reveals a conformational rearrangement in the K48-linked diubiquitin substrate.
abstract: Deubiquitination has emerged as an essential regulatory mechanism of a number of cellular processes. An in-depth understanding of deubiquitinating enzyme (DUB) catalysis, particularly the mode of ubiquitin binding and the individual steps in the DUB catalytic turnover, is imperative for exploiting DUBs for therapeutic intervention. In this work, we present a transient kinetic study of USP2 in hydrolyzing a model substrate Ub-AMC and a physiological substrate K48-linked diubiquitin. We conducted stopped-flow fluorescence analyses of the binding of mono- and diubiquitin to an inactive USP2 mutant and unveiled interesting differences in the binding kinetics between the two substrates. While a simple one-step binding of monoubiquitin to USP2 was observed, a biphasic binding was evident for diubiquitin. We further followed the deubiquitination reaction of Ub-AMC and K48-linked IQF-diubiquitin by USP2 using stopped-flow florescence under a single-turnover condition. Global fitting of the reaction traces revealed differences in the microscopic rate constants between Ub-AMC and the physiological diubiquitin substrate. Our binding and single-turnover data support a conformational rearrangement of the diubiquitin substrate in USP2-catalyzed deubiquitination. This finding is significant given the recent finding that the K48-linked diubiquitin is dynamic in its conformation. Our results provide useful insights into the mechanism of how USP recognizes ubiquitin moieties in a chain structure, which is important for understanding USP catalysis and developing inhibitors against USPs.
authors.affiliation: Department of Chemistry and Biochemistry, 214A Drake Hall, University of Delaware, Newark, DE 19716, USA.
authors.forename: William P
authors.initials: WP
authors.lastname: Bozza
authors.forename: Qin
authors.initials: Q
authors.lastname: Liang
authors.forename: Ping
authors.initials: P
authors.lastname: Gong
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Zhuang
mesh_terms: D003374:Coumarins / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011487:Protein Conformation; D043222:Ubiquitin Thiolesterase; D014452:Ubiquitins / Q000378:metabolism*
chemical_list: D003374:Coumarins; C109184:UBD protein, human; D014452:Ubiquitins; C110770:ubiquitin C-terminal 7-amido-4-methylcoumarin; D010450:Endopeptidases; C108665:USP2 protein, human; D043222:Ubiquitin Thiolesterase