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Updated: Jun 17, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
Kinetics of the transfer of ubiquitin from UbcH7 to E6AP
Carrie Purbeck1, Ziad M Eletr, Brian Kuhlman
1Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
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.
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