Allosteric regulation of a ribozyme activity through ligand-induced conformational change
Allosteric regulation of a ribozyme activity through ligand-induced conformational change
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DOI：
10.1093/nar/26.14.3379
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发表时间：
1998-07-15
影响因子：
14.9
通讯作者：
Sugiura, Y
中科院分区：
文献类型：
--
作者：
Araki, M;Okuno, Y;Sugiura, Y
以锤头状核酶为活性位点，黄素特异性RNA适体为调控位点，设计了一种变构核酶。我们构建了六个变体，在连接区（茎II）中具有一系列碱基对。在单转换条件下，在不存在和存在黄素单甘肽（FMN）的情况下进行动力学研究。有趣的是，FMN的添加不影响具有5-6 bp接头的构建体的切割速率，但刺激具有较短接头的那些的催化活性。特别地，在加入饱和量的FMN后，Rz 3的表观k（cat）增加了接近10倍。为了确定速率常数（K-m和k（cat）），进一步研究了由FMN最有效调节的核酶。FMN主要影响的k（猫）值，反映了速率限制的整体裂解反应的构象变化步骤，取决于在茎II的螺旋形成。可能，FMN的影响，通过茎II形成的裂解反应所需的结构的取向。化学修饰的结果表明，FMN的适体结构域的结合诱导茎II的催化活性所需的螺旋形成。因此，一个特定的FMN介导的变构相互作用似乎促进了茎II中从开放结构到封闭结构的构象改变。变构效应中构象修饰的概念与其他变构酶一致，表明这种构象变化是生物系统中变构酶的基本特征。
An allosteric ribozyme has been designed using the hammerhead ribozyme as the active site and a flavin-specific RNA aptamer as a regulatory site. We constructed six variants with a series of base pairs in the linker region (stem II). Under single turnover conditions, kinetic studies were carried out in the absence and presence of flavin mononucleotide (FMN), Interestingly, FMN addition did not influence the cleavage rate of constructs with a 5-6 bp linker but stimulated the catalytic activity of those bearing a shorter linker. In particular, the apparent k(cat) of Rz3 increases by similar to 10-fold upon addition of saturating amounts of FMN, To determine the rate constants (K-m, and k(cat)), the ribozyme regulated most effectively by FMN was further investigated. FMN mainly affected the k(cat) value, reflecting the rate limiting conformational change step of the overall cleavage reaction, depending on helix formation in stem II. Probably, FMN influences the orientation of structures necessary for the cleavage reaction through stem II formation. The result of chemical modification revealed that binding of FMN to the aptamer domain induced the helix formation in stem II required for catalytic activity. Therefore, a specific FMN-mediated allosteric interaction seems to promote a conformational alteration from an open to a closed structure in stem II. The concept of conformational modification in the allosteric effect is consistent with other allosteric enzymes, suggesting that such a conformational change is a fundamental feature of allosteric enzymes in biological systems.