Ubiquitination Of Substrates By E6ap/Ube3a Ligase

Only a narrow range of E6AP ubiquitin ligase activity is allowed for normal neural development. Enzyme activation by Ube3A gene duplication is linked with familial autism spectrum disorder while loss of function results in the Angelman syndrome neurological disorder. BIOGRID recognizes 174 E6AP-interactors but only a few have been validated as substrates for ubiquitination. Recently, phosphorylation on E6APT485, an autism related residue, affected RPN10/S5A substrate ubiquitination in cells. The current aim included kinetic analysis of E6AP-catalyzed conjugation of target proteins to provide new insights into the mechanism of substrate ubiquitination. E6AP-dependent Lys48-polyubiquitin chain assembly in the absence of substrate requires two functionally distinct UbcH7∼ubiquitin binding sites on the ligase surface and oligomerization. Here, the E6AP ubiquitin-function was analyzed in the presence of RNP10/S5A (regulatory subunit of proteasome 26S) and PRDX1 (antioxidant enzyme). Rates of substrate ubiquitin adduct formation were analyzed under E6AP rate-limiting conditions. Adducts of PRDX1-Ub1 or RPN10/S5A-Ub1 showed Km values of 8±1 μM and 0.2±0.06 μM, respectively; while the kcat values were 0.3 s−1, comparable to 0.5 s−1 observed for polyubiquitin chain assembly without substrate and indicating that the ligase cannot distinguish the lysine nucleophile in Lys48-ubiquitin and Lys-target protein ubiquitination. Analysis of pH-dependent E6AP ligase function inferred a pKa of ∼8.4, either in the absence or presence of PRDX1. Removal of the first 250 N-terminal residues reduced ubiquitination of both substrates supporting the presence of previously unrecognized substrate binding domains in this region. A T485D mutation mimicking E6AP phosphorylation, or a D212A mutation, an Angelman syndrome mutation, abrogated substrate ubiquitination, although they retained the polyubiquitin chain assembly function. The results provide new insights of the E6AP ubiquitination mechanism in the presence of target proteins that might explain the deleterious effect of some mutations associated with Angelman syndrome.