Kinetics of the Transfer of Ubiquitin from UbcH7 to E6AP

Prior to substrate ubiquitination by HECT-E3 ligases, ubiquitin must first be activated by El and then transferred via a series of transthiolation reactions from El 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 similar to 2.3 x 10(5) M-1 s(-1). The measured parameters place limits on substrate-E6AP binding lifetimes required for processive polyubiquitination.