Wu Pei-Ying, Hanlon Mary, Eddins Michael, Tsui Colleen, Rogers Richard S, Jensen Jane P, Matunis Michael J, Weissman Allan M, Wolberger Cynthia, Pickart Cecile M
Department of Biochemistry and Molecular Biology/Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205, USA.
EMBO J. 2003 Oct 1;22(19):5241-50. doi: 10.1093/emboj/cdg501.
Ubiquitin (Ub) regulates diverse functions in eukaryotes through its attachment to other proteins. The defining step in this protein modification pathway is the attack of a substrate lysine residue on Ub bound through its C-terminus to the active site cysteine residue of a Ub-conjugating enzyme (E2) or certain Ub ligases (E3s). So far, these E2 and E3 cysteine residues are the only enzyme groups known to participate in the catalysis of conjugation. Here we show that a strictly conserved E2 asparagine residue is critical for catalysis of E2- and E2/RING E3-dependent isopeptide bond formation, but dispensable for upstream and downstream reactions of Ub thiol ester formation. In contrast, the strictly conserved histidine and proline residues immediately upstream of the asparagine are dispensable for catalysis of isopeptide bond formation. We propose that the conserved asparagine side chain stabilizes the oxyanion intermediate formed during lysine attack. The E2 asparagine is the first non-covalent catalytic group to be proposed in any Ub conjugation factor.
泛素（Ub）通过与其他蛋白质结合来调节真核生物中的多种功能。这种蛋白质修饰途径的决定性步骤是底物赖氨酸残基攻击与泛素结合酶（E2）或某些泛素连接酶（E3）的活性位点半胱氨酸残基通过其C末端相连的泛素。到目前为止，这些E2和E3半胱氨酸残基是已知参与共轭催化的唯一酶基团。在这里，我们表明一个严格保守的E2天冬酰胺残基对于E2和E2/RING E3依赖性异肽键形成的催化至关重要，但对于泛素硫酯形成的上游和下游反应是可有可无的。相比之下，天冬酰胺上游紧邻的严格保守的组氨酸和脯氨酸残基对于异肽键形成的催化是可有可无的。我们提出保守的天冬酰胺侧链稳定了赖氨酸攻击过程中形成的氧阴离子中间体。E2天冬酰胺是在任何泛素共轭因子中提出的第一个非共价催化基团。
