title: Kinetics of the transfer of ubiquitin from UbcH7 to E6AP.
abstract: Prior to substrate ubiquitination by HECT-E3 ligases, ubiquitin must first be activated by E1 and then transferred via a series of transthiolation reactions from E1 to E2 and from E2 to E3. We have measured the rate constants and binding affinities underlying the transfer of ubiquitin from E2 UbcH7 to the HECT domain of E3 E6AP. We show that charged UbcH7 and free UbcH7 bind E6AP with similar affinities and that at 37 degrees C the second-order rate constant for the reaction (k(cat)/K(m)) equals approximately 2.3 x 10(5) M(-1) s(-1). The measured parameters place limits on substrate-E6AP binding lifetimes required for processive polyubiquitination.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
authors.forename: Carrie
authors.initials: C
authors.lastname: Purbeck
authors.forename: Ziad M
authors.initials: ZM
authors.lastname: Eletr
authors.forename: Brian
authors.initials: B
authors.lastname: Kuhlman
mesh_terms: D001667:Binding, Competitive; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D021381:Protein Transport; D055672:Static Electricity; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry* / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; C471639:UBE3A protein, human; D044767:Ubiquitin-Protein Ligases