Mechanism of ubiquitin conjugating enzyme E2-230K: Catalysis involving a thiol relay
Mechanism of ubiquitin conjugating enzyme E2-230K: Catalysis involving a thiol relay
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DOI：
10.1021/bi952105y
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发表时间：
1996-02-06
期刊：
影响因子：
2.9
通讯作者：
Pickart, CM
中科院分区：
文献类型：
--
作者：
Berleth, ES;Pickart, CM
泛素与胞内蛋白的共价结合是26S酶降解的信号。结合通常是通过激活(EL)、结合(E2)和连接酶(E3)的顺序作用来完成的。这些酶中的每一个都形成了与泛素共价的硫醇酯，作为其催化循环的一部分。在大多数情况下，泛素结合酶(E2)的明显作用是将泛素从EL活性部位转移到E3活性部位。然后，泛素从E3传递到底物赖氨酸残基。一种异常大的网织红细胞特异性酶，称为E2-230K，在E2酶的大家族中是独一无二的，它容易受到无机亚砷酸盐的抑制[Klemperer等人。(1989)生物化学28,6035-6041]，我们发现苯基氧化砷有效地抑制E2-230K，显然是通过与酶的邻近半胱氨酸残基结合：结合的氨基苯基氧化亚胺(NEM)部分保护酶不被N-乙基马来酰亚胺(NEM)灭活，而预先用NEM灭活阻止了酶与固定化苯基氧化亚胺的结合。对抑制机制的研究表明，完全抑制稳态周转的(氨基苯基)氧化亚砷浓度对预先形成的E_2-泛素加合物的周转没有影响。然而，当该酶在加合物形成开始前与该浓度的抑制剂预先孵育时，与E2相关的泛素水平降低了60%。这些结果与酶的两个半胱氨酸残基依次与泛素形成硫醇酯的模型一致，其中第二个半胱氨酸残基与酶-抑制剂复合体中的砷结合。在该模型中，E2-230K作为E2-E3的杂交体发挥作用。
Covalent conjugation of ubiquitin to intracellular proteins is a signal for degradation by the 26S protease. Conjugation is usually accomplished by the sequential action of activating (El), conjugating (E2), and ligase (E3) enzymes. Each of these enzymes forms a covalent thiol ester with ubiquitin as part of its catalytic cycle. In most cases, the apparent role of the ubiquitin conjugating enzyme (E2) is to transfer ubiquitin from the El active site to the E3 active site. Ubiquitin is then delivered from E3 to the substrate lysine residue. An unusually large, reticulocyte-specific enzyme, known as E2-230K, is unique among the large family of E2 enzymes is being susceptible to inhibition by inorganic arsenite [Klemperer et al. (1989) Biochemistry 28, 6035-6041], We show that phenylarsenoxides potently inhibit E2-230K, apparently by binding to vicinal Cys residues of the enzyme: bound aminophenylarsenoxide partially protects the enzyme against inactivation by N-ethylmaleimide (NEM), and prior enzyme inactivation with NEM blocks enzyme binding to immobilized phenylarsenoxide. Studies on the mechanistic basis of inhibition showed that a concentration of (aminophenyl)arsenoxide that produced complete inhibition of steady-state turnover had no effect on the turnover of the preformed E2-ubiquitin adduct. However, when the enzyme was preincubated with this concentration of inhibitor prior to initiation of adduct formation, the level of E2-associated ubiquitin was reduced by 60%. These results are consistent with a model in which two Cys residues of the enzyme sequentially form thiol esters with ubiquitin and the second of these Cys residues is bound to arsenic in the enzyme-inhibitor complex. In this model, E2-230K functions as an E2-E3 hybrid.