Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation.
Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation.
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DOI：
10.1016/s0021-9258(18)34958-5
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发表时间：
1982-03
期刊：
影响因子：
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通讯作者：
A. Haas;J. V. Warms;A. Hershko;I. A. Rose
中科院分区：
文献类型：
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作者：
A. Haas;J. V. Warms;A. Hershko;I. A. Rose
兔网织红细胞裂解物中的能量依赖性蛋白水解对ATP和泛素都有绝对的需求，这被认为是由于泛素（Ub）与底物蛋白的ATP依赖性共价缀合作为信号事件。最近，负责激活泛素的酶已被纯化到近同质的共价亲和层析。在目前的工作中，我们证明了酶催化净反应（公式：见正文），其中既形成了酶结合的COOH-末端泛素硫酯和COOH-末端泛素腺苷酸。该复合物足够稳定，可以通过排阻色谱法分离，并且含有比率为2的泛素/AMP。两种形式的共价连接的泛素的存在下提供了一个观察泛素依赖的ATP：PPi和ATP：AMP交换的基本原理。巯基位点与碘乙酰胺的选择性衍生化导致修饰的酶仅能够催化ATP：PPi交换并仅形成泛素腺苷酸。巯基位点的衍生化防止在重构系统中形成缀合物，表明泛素硫酯是随后缀合物形成的近端供体。一个三步骤的机制，提出了泛素活化的酶之前的共轭。
Energy-dependent proteolysis in rabbit reticulocyte lysates has an absolute requirement for both ATP and ubiquitin that is believed due to the ATP-dependent covalent conjugation of ubiquitin (Ub) to substrate proteins as a signal event. Recently the enzyme responsible for activation of ubiquitin has been purified to near homogeneity by covalent affinity chromatography. In the present work we demonstrate that the enzyme catalyzes the net reaction (formula: see text); in which both an enzyme-bound COOH-terminal ubiquitin thiolester and a COOH-terminal ubiquitin adenylate are formed. The complex is sufficiently stable to be isolated by exclusion chromatography and contains ubiquitin/AMP in a ratio of 2. The presence of two forms of covalently linked ubiquitin provides a rationale for the observation of both ubiquitin-dependent ATP:PPi and ATP:AMP exchange. Selective derivatization of the thiol site with iodoacetamide results in a modified enzyme capable of catalyzing only ATP:PPi exchange and in forming only ubiquitin adenylate. Derivatization of the thiol site prevents conjugate formation in a reconstituted system, demonstrating that the ubiquitin thiolester is the proximal donor for subsequent conjugate formation. A three-step mechanism is proposed for ubiquitin activation by the enzyme prior to conjugation.