Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Biochemistry. 2010 Feb 23;49(7):1361-3. doi: 10.1021/bi9014693.
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.
在 HECT-E3 连接酶将底物泛素化之前，泛素必须首先被 E1 激活，然后通过一系列转硫反应从 E1 转移到 E2，再从 E2 转移到 E3。我们已经测量了将泛素从 E2 UbcH7 转移到 E3 E6AP 的 HECT 结构域的反应中的速率常数和结合亲和力。我们表明，带电荷的 UbcH7 和游离的 UbcH7 与 E6AP 具有相似的亲和力，并且在 37°C 下，反应的二级速率常数（kcat/Km）约等于 2.3×10(5) M(-1) s(-1)。所测量的参数限制了连续多泛素化所需的底物-E6AP 结合寿命。
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