ArticleThe Journal of biological chemistry2013
E6AP/UBE3A ubiquitin ligase harbors two E2~ubiquitin binding sites.
Article in The Journal of biological chemistry, 2013. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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Who cites it
29 citing papers in PubMed, 41 citations in OpenAlex.
- HECT-type ubiquitin ligases: Emerging principles in the era of full-length structures.The Journal of biological chemistry · 2026Review
- Chemical Tools for Probing the Ub/Ubl Conjugation Cascades.Chembiochem : a European journal of chemical biology · 2025Review
- Article
- Redefining the catalytic HECT domain boundaries for the HECT E3 ubiquitin ligase family.Bioscience reports · 2022Article
- Review
- An Angelman syndrome substitution in the HECT E3 ubiquitin ligase C-terminal Lobe of E6AP affects protein stability and activity.PloS one · 2020Article
- Stepwise multipolyubiquitination of p53 by the E6AP-E6 ubiquitin ligase complex.The Journal of biological chemistry · 2019Article
- Loss of nuclear UBE3A causes electrophysiological and behavioral deficits in mice and is associated with Angelman syndrome.Nature neuroscience · 2019Article
- Analysis of ubiquitin recognition by the HECT ligase E6AP provides insight into its linkage specificity.The Journal of biological chemistry · 2019Article
- Enzymatic Logic of Ubiquitin Chain Assembly.Frontiers in physiology · 2019Review
- A patent review of the ubiquitin ligase system: 2015-2018.Expert opinion on therapeutic patents · 2018Review
- Oligomerization of the HECT ubiquitin ligase NEDD4-2/NEDD4L is essential for polyubiquitin chain assembly.The Journal of biological chemistry · 2018Article
- Developing Small-Molecule Inhibitors of HECT-Type Ubiquitin Ligases for Therapeutic Applications: Challenges and Opportunities.Chembiochem : a European journal of chemical biology · 2018Review
- The mechanism of neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2)/NEDD4L-catalyzed polyubiquitin chain assembly.The Journal of biological chemistry · 2017Article
- Article
- Mechanism of ubiquitin chain synthesis employed by a HECT domain ubiquitin ligase.The Journal of biological chemistry · 2017Article
- Mechanism and disease association of E2-conjugating enzymes: lessons from UBE2T and UBE2L3.The Biochemical journal · 2016Review
- Structural insights into the catalysis and regulation of E3 ubiquitin ligases.Nature reviews. Molecular cell biology · 2016Review
- System-Wide Modulation of HECT E3 Ligases with Selective Ubiquitin Variant Probes.Molecular cell · 2016Article
- Review
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3 authors at 1 institution in 1 country.
Funding
Abstract
By exploiting (125)I-polyubiquitin chain formation as a functional readout of enzyme activity, we have quantitatively examined the mechanism of human E6AP/UBE3A for the first time. Initial rate studies identify UbcH7 as the cognate E2 carrier protein for E6AP, although related Ubc5 isoforms and the ISG15-specific UbcH8 paralog also support E6AP with reduced efficacy due to impaired binding and catalytic competence. Initial rates of polyubiquitin chain formation displayed hyperbolic kinetics with respect to UbcH7 concentration (K(m) = 57.6 ± 5.7 nM and kcat = 0.032 ± 0.001 s(-1)) and substrate inhibition above 2 μM. Competitive inhibition by an isosteric UbcH7C86S-ubiquitin oxyester substrate analog (K(i) = 64 ± 18 nM) demonstrates that Km reflects intrinsic substrate affinity. In contrast, noncompetitive inhibition by a UbcH7C86A product analog (K(i) = 7 ± 0.7 μM) and substrate inhibition at high concentrations require two functionally distinct E2∼ubiquitin substrate binding sites. The kinetics of polyubiquitin chain formation reflect binding at a cryptic Site 1 not previously recognized that catalyzes E6AP∼ubiquitin thioester formation. Subsequent binding of E2∼ubiquitin at the canonical Site 2 present in the extant crystal structure is responsible for polyubiquitin chain elongation. Other rate studies show that the conserved -4 Phe(849) residue is required for polyubiquitin chain formation rather than target protein conjugation as originally suggested. The present studies unambiguously preclude earlier models for the mechanism of Hect domain-catalyzed conjugation through the canonical binding site suggested by the crystal structure and define a novel two-step mechanism for formation of the polyubiquitin degradation signal.
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