Substrate specificity of deubiquitinating enzymes: Ubiquitin C-terminal hydrolases
Substrate specificity of deubiquitinating enzymes: Ubiquitin C-terminal hydrolases
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DOI：
10.1021/bi972274d
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发表时间：
1998-03-10
期刊：
影响因子：
2.9
通讯作者：
Wilkinson, KD
中科院分区：
文献类型：
--
作者：
Larsen, CN;Krantz, BA;Wilkinson, KD
泛素C末端水解酶（UGH）是水解泛素C末端酯和酰胺的去泛素化酶。在这里，我们报告的两个人类UCH同工酶（同工酶L1和L3）的泛素衍生物的加工数量，并发现这些酶的P1'氨基酸的基础上显示很少的歧视，除了脯氨酸被切割缓慢。通过α-或ε-氨基连接的泛肽基赖氨酸衍生物以相同的速率水解。只有当离去基团较大时，同工酶特异性水解偏好才明显。泛素基因产物可被一种或两种UCH同工酶协同加工，纯化的UbCEP 52可被UCH同工酶L3水解。UbCEP 52与核酸的结合将其转化为对这些酶的加工具有抗性的形式，显然是因为形成了更大、更紧密折叠的底物。与该假设一致的是观察到这些酶不水解大的泛素衍生物，如N-α-泛素基-细胞色素-c、N-β-（K48）聚泛素基-溶菌酶或N-α-泛素基-β-半乳糖苷酶融合蛋白。因此，这些酶快速且优先地从泛素的C-末端切割小的离去基团如氨基酸和寡肽，但不切割较大的离去基团如蛋白质。这些数据表明，UCH的生理作用是水解泛素的小加合物，并产生游离的单体泛素从泛素前蛋白，但不去泛素化的泛素蛋白缀合物或拆卸聚泛素链。
Ubiquitin C-terminal hydrolases (UGH) are deubiquitinating enzymes which hydrolyze C-terminal esters and amides of ubiquitin. Here we report the processing of a number of ubiquitin derivatives by two human UCH isozymes (isozymes L1 and L3) and find that these enzymes show little discrimination based on the P1' amino acid, except that proline is cleaved slowly. Ubiquitinyllysine derivatives linked by the alpha- or epsilon-amino group are hydrolyzed at identical rates. Isozyme-specific hydrolytic preferences are only evident when the leaving group is large. The ubiquitin gene products can be cotranslationally processed by one or both of these UCH isozymes, and purified UbCEP52 can be hydrolyzed by UCH isozyme L3. Binding of nucleic acid by UbCEP52 converts it to a form resistant to processing by these enzymes, apparently because of the formation of a larger, more tightly folded substrate. Consistent with this postulate is the observation that these enzymes do not hydrolyze large ubiquitin derivatives such as N-epsilon-ubiquitinyl-cytochrome-c, N-epsilon-(K48)polyubiquitinyl-lysozyme, or an N-alpha-ubiquitinyl-beta-galactosidase fusion protein. Thus, these enzymes rapidly and preferentially cleave small leaving groups such as amino acids and oligopeptides from the C-terminus of ubiquitin, but not larger leaving groups such as proteins. These data suggest that the physiological role of UCH is to hydrolyze small adducts of ubiquitin and to generate free monomeric ubiquitin from ubiquitin proproteins, but not to deubiquitinate ubiquitin-protein conjugates or disassemble polyubiquitin chains.