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首页> 外文期刊>International Journal of Biological Macromolecules: Structure, Function and Interactions >Cooperativity in the two-domain arginine kinase from the sea anemone Anthopleura japonicus. II. Evidence from site-directed mutagenesis studies
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Cooperativity in the two-domain arginine kinase from the sea anemone Anthopleura japonicus. II. Evidence from site-directed mutagenesis studies

机译：来自海葵Anthopleura japonicus的两个域精氨酸激酶的协同作用。二。定点诱变研究的证据

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摘要

The arginine kinase (AK) from the sea anemone Anthopleura japonicus has an unusual two-domain structure (contiguous dimer; denoted by D1-D2). In a previous report, we suggested cooperativity in the contiguous dimer, which may be a result of domain-domain interactions, using MBP-fused enzymes. To further understand this observation, we inserted six-Lys residues into the linker region of the two-domain AK (D1-K6-D2 mutant) using His-tagged enzyme. The dissociation constants, K_a and K_(ia), of the mutant were similar to those of the wild-type enzyme but the catalytic constant, k_(cat), was decreased to 28% that of the wild-type, indicating that some of the domain-domain interactions are lost due to the six-Lys insertion. Y68 plays a major role in arginine binding in the catalytic pocket in Limulus AK, and introduction of mutation at the Y68 position virtually abolishes catalytic activity. Thus, the constructed D1(Y68G)-D2 and D1-D2(Y68G) mutants mimic the D1(inactive)-D2(active) and D1(active)-D2(inactive) enzymes, respectively. The k_(cat) values of both Y68 mutants were decreased to 13-18% that of the wild-type enzyme, which is much less than the 50% level of the two-domain enzyme. Thus, it is clear that substrate-binding to both domains is necessary for full expression of activity. In other words, substrate-binding appears to act as the trigger of the functional cooperativity in two-domain AK.
机译：来自海葵Anthopleura japonicus的精氨酸激酶（AK）具有不同寻常的两个结构域结构（连续的二聚体;用D1-D2表示）。在以前的报告中，我们建议使用MBP融合酶在连续二聚体中进行协同操作，这可能是域-域相互作用的结果。为了进一步了解该观察，我们使用His-tagged酶将6-Lys残基插入到两个结构域AK（D1-K6-D2突变体）的接头区域中。突变体的解离常数K_a和K_（ia）与野生型酶的解离常数相似，但催化常数k_（cat）降至野生型酶的28％，表明其中一些由于插入了6个Lys，域之间的相互作用会丢失。 Y68在Li AK的催化口袋中的精氨酸结合中起主要作用，并且在Y68位置引入突变实际上消除了催化活性。因此，构建的D1（Y68G）-D2和D1-D2（Y68G）突变体分别模拟D1（非活性）-D2（活性）和D1（活性）-D2（非活性）酶。两个Y68突变体的k_（cat）值均降低至野生型酶的k_（cat）值，远低于两结构域酶的50％水平。因此，很清楚，底物与两个结构域的结合对于活性的充分表达是必需的。换句话说，底物结合似乎充当了两个域AK中功能合作性的触发因素。

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