STRUCTURE AND FUNCTION OF UBIQUITIN - EVIDENCE FOR DIFFERENTIAL INTERACTIONS OF ARGININE-74 WITH THE ACTIVATING ENZYME AND THE PROTEASES OF ATP-DEPENDENT PROTEOLYSIS
STRUCTURE AND FUNCTION OF UBIQUITIN - EVIDENCE FOR DIFFERENTIAL INTERACTIONS OF ARGININE-74 WITH THE ACTIVATING ENZYME AND THE PROTEASES OF ATP-DEPENDENT PROTEOLYSIS
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DOI：
10.1021/bi00396a019
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发表时间：
1987-11-03
期刊：
影响因子：
2.9
通讯作者：
WILKINSON, KD
中科院分区：
文献类型：
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作者：
DUERKSENHUGHES, PJ;XU, XX;WILKINSON, KD
用阴离子型、精氨酸特异性试剂4-（氧代乙酰基）苯氧基乙酸修饰泛素，以研究分子的结构与功能之间的关系。通过阴离子交换高效液相色谱法从反应混合物中纯化四种不同的衍生物（A、B、C和D），并进行胰蛋白酶肽图谱分析以确定修饰的位置。这些衍生物在纯化、胰蛋白酶水解和肽图谱分析所需的整个过程中是稳定的。衍生物A在精氨酸-42处被修饰，衍生物B在精氨酸-72处被修饰，衍生物C在精氨酸-42和-72处被修饰，衍生物D在精氨酸-74处被修饰。用14 C标记的4-（氧代乙酰基）苯氧基乙酸修饰泛素表明，该试剂与蛋白质的精氨酸残基形成稳定的1：1复合物。测试了天然泛素和四种衍生物中的每一种刺激ATP和焦磷酸之间的32 P交换的能力，这是由泛素依赖性蛋白水解途径的酶1催化的反应。A和C能够促进这种交换，其速率仅为天然泛素的15%，B刺激交换至天然水平的25%，而D刺激交换至天然水平的60%。没有一个衍生物能够促进显着水平的泛素依赖性蛋白水解。D能够与外源性和内源性蛋白质形成缀合物，其程度与天然遍在蛋白非常相似，这表明它无法刺激遍在蛋白依赖性蛋白水解是由于缀合物形成步骤之外的缺陷造成的。这些结果表明，在该系统中，泛素-42，-72，和-74的完整性是必需的泛素的完整功能，并建议泛素活化酶（E1）和系统的蛋白酶识别不同的区域或构象的泛素。
Ubiquitin was modified with the anionic, arginine-specific reagent 4-(oxoacetyl)phenoxyacetic acid in order to study the relationship between structure and function of the molecule. Four different derivatives (A, B, C, and D) were purified from the reaction mixture by anion-exchange high-performance liquid chromatography and subjected to tryptic peptide mapping to determine the location of the modification(s). These derivatives were stable throughout the procedures required for purification, tryptic hydrolysis, and peptide mapping. Derivative A was modified at arginine-42, derivative B at arginine-72, derivative C at arginines-42 and -72, and derivative D at arginine-74. Modification of ubiquitin with 14C-labeled 4-(oxoacetyl)phenoxyacetic acid indicated that the reagent formed a stable; 1:1 complex with arginine residues of the protein. Native ubiquitin and each of the four derivatives were tested for their ability to stimulate 32P exchange beteen ATP and pyrophosphate, a reaction catalyzed by enzyme 1 of the ubiquitin-dependent proteolytic pathway. A and C were capable of promoting this exchange at a rate only 15% that of native ubiquitin, B stimulated the exchange to 25%, and D stimulated exchange to 60% of the native level. None of the derivatives was capable of promoting a significant level of ubiquitin-dependent proteolysis. D was capable of forming conjugates with exogenous and endogenous proteins to an extent very similar to that of native ubiquitin, suggesting that its inability to stimulate ubiquitin-dependent proteolysis was due to a defect in a step beyond that of conjugate formation. These results indicate that in this system, the integrity of arginines-42, -72, and -74 is essential for full function of ubiquitin and suggest that the ubiquitin activating enzyme (E1) and the protease(s) of the system recognize different regions or conformations of ubiquitin.