title: Bi-substrate kinetic analysis of an E3-ligase-dependent ubiquitylation reaction.
abstract: Little is known about the kinetic mechanism of E3 ubiquitin ligases. This work describes basic methodology to investigate the kinetic mechanism of E3 ubiquitin ligases. The method used steady state, bi-substrate kinetic analysis of an E3 ligase-catalyzed monoubiquitylation reaction using ubiquitin-conjugated E2 (E2ub) and a mutant IkappaBalpha as substrates to evaluate whether the E3-catalyzed ubiquitin transfer from E2ub to protein substrate was sequential, meaning both substrates bound before products leaving, or ping pong, meaning that ubiquitin-conjugated E2 would bind, transfer ubiquitin to the E3, and debind before binding of protein substrate. The method requires the E3 reaction to be rate limiting and at steady state. This was accomplished through optimization of the conditions to ensure that the E3-dependent transfer of ubiquitin from E2ub to substrate was rate limiting. We observed a sequential bi-substrate E3-dependent ubiquitylation reaction on using E2UBCH7 and IkappaBalphaSS32/36EE (IkappaBalphaee as substrates and a partially purified Jurkat cell lysate as a source for the E3 ligase activity). The sequential bi-substrate kinetic mechanism is consistent with the formation of a ternary complex among E2UBCH7, IkappaBalphaSS32/36EE, and E3 before the transfer of ubiquitin from E2UBCH7 to IkappaBalphaSS32/36EE. The described method should be of use to characterize the kinetic mechanism of other E3 ligase-catalyzed ubiquitylation reactions.
authors.affiliation: Biochemical Pharmacology, Roche Bioscience, Palo Alto, California, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Swinney
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Rose
authors.forename: Amy Y
authors.initials: AY
authors.lastname: Mak
authors.forename: Ina
authors.initials: I
authors.lastname: Lee
authors.forename: Liliana
authors.initials: L
authors.lastname: Scarafia
authors.forename: Yi-Zheng
authors.initials: YZ
authors.lastname: Xu
mesh_terms: D007700:Kinetics; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*
chemical_list: D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases