A HYBRID OF BOVINE PANCREATIC RIBONUCLEASE AND HUMAN ANGIOGENIN - AN EXTERNAL LOOP AS A MODULE CONTROLLING SUBSTRATE-SPECIFICITY
A HYBRID OF BOVINE PANCREATIC RIBONUCLEASE AND HUMAN ANGIOGENIN - AN EXTERNAL LOOP AS A MODULE CONTROLLING SUBSTRATE-SPECIFICITY
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DOI：
10.1093/protein/4.7.831
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发表时间：
1991-10-01
期刊：
影响因子：
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通讯作者：
BENNER, SA
中科院分区：
文献类型：
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作者：
ALLEMANN, RK;PRESNELL, SR;BENNER, SA
三个同源核糖核酸酶（核糖核酸酶A，血管生成素和牛精液核糖核酸酶）的序列的比较确定了三个表面环，这三个蛋白质之间的高度可变。对比了两种假设：（i）这种变异可能是三种蛋白质不同催化活性的原因;（ii）这种变异只是表面环在序列上经历快速中性趋异的一个例子。制备了血管生成素和牛胰腺核糖核酸酶（RNase）A的三种杂交物，其中这些环中取自血管生成素的区域插入RNase A。三种杂化物中有两种具有不显著的催化性能。然而，含有血管生成素残基63-74的RNase A突变体对尿苷酰-（3' -> 5'）-腺苷（UpA）的催化活性大大降低，而作为体外翻译抑制剂的催化活性略有增加。这两种催化行为都是血管生成素的特征。这是蛋白质中工程化外部环的第一个例子之一。此外，这些结果与最近从补充实验中获得的结果是互补的，其中RNA酶的残基59-70被插入血管生成素中[哈珀和Vallee（1989）Biochemistry，28，1875-1884]。因此，RNase A残基63-74中的外环似乎至少部分地表现为可互换的“模块”，其以与蛋白质中其他区域的影响隔离的方式影响酶中的底物特异性。
A comparison of the sequences of three homologous ribonucleases (RNase A, angiogenin and bovine seminal RNase) identifies three surface loops that are highly variable between the three proteins. Two hypotheses were contrasted: (i) that this variation might be responsible for the different catalytic activities of the three proteins; and (ii) that this variation is simply an example of surface loops undergoing rapid neutral divergence in sequence. Three hybrids of angiogenin and bovine pancreatic ribonuclease (RNase) A were prepared where regions in these loops taken from angiogenin were inserted into RNase A. Two of the three hybrids had unremarkable catalytic properties. However, the RNase A mutant containing residues 63-74 of angiogenin had greatly diminished catalytic activity against uridylyl-(3' --> 5')-adenosine (UpA), and slightly increased catalytic activity as an inhibitor of translation in vitro. Both catalytic behaviors are characteristic of angiogenin. This is one of the first examples of an engineered external loop in a protein. Further, these results are complementary to those recently obtained from the complementary experiment, where residues 59-70 of RNase were inserted into angiogenin [Harper and Vallee (1989) Biochemistry, 28, 1875-1884]. Thus, the external loop in residues 63-74 of RNase A appears to behave, at least in part, as an interchangeable 'module' that influences substrate specificity in an enzyme in a way that is isolated from the influences of other regions in the protein.