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首页> 外文期刊>Journal of Molecular Biology >Functional Group Requirements in the Probable Active Site of the VS Ribozyme.
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Functional Group Requirements in the Probable Active Site of the VS Ribozyme.

机译：VS核酶可能活跃位点中的功能组要求。

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摘要

The VS ribozyme catalyses the site-specific cleavage of a phosphodiester linkage by a transesterification reaction that entails the attack of the neighbouring 2'-oxygen with departure of the 5'-oxygen. We have previously suggested that the A730 loop is an important component of the active site of the ribozyme, and that A756 is especially important in the cleavage reaction. Functional group modification experiments reported here indicate that the base of A756 is more important than its ribose for catalysis. A number of changes to the base, including complete ablation, lead to cleavage rates that are reduced 1000-fold, while removal of the 2'-hydroxyl group from the ribose results in tenfold slower cleavage. 2-Aminopurine fluorescence experiments indicate that this 2'-hydroxyl group is important for the structure of the A730 loop. Catalytic activity is especially sensitive to changes involving the exocyclic amine of A756; by contrast, the cleavage activity is only weakly sensitive to modification at the 7-position of the purine nucleus. These results suggest that the Watson-Crick edge of the adenine base is important in ribozyme function. We sought to test the possibility of a direct role of the nucleobase in the chemistry of the cleavage reaction. Addition of imidazole base in the medium failed to restore the activity of a ribozyme from which the nucleobase of A756 was removed. However, no restoration was obtained with exogenous adenine base either, indicating that the cavity that might result from ablation of the base was closed.
机译：VS核酶通过酯交换反应催化磷酸二酯键的位点特异性切割，该酯交换反应伴随着5'-氧的离开而攻击相邻的2'-氧。我们以前曾提出，A730环是核酶活性位点的重要组成部分，而A756在裂解反应中尤其重要。此处报道的官能团修饰实验表明，对于催化作用，A756的碱基比其核糖更重要。碱基的许多变化（包括完全消融）导致切割速率降低了1000倍，而从核糖中去除2'-羟基会使切割速率减慢十倍。 2-氨基嘌呤荧光实验表明，该2'-羟基对于A730环的结构很重要。催化活性对涉及A756环外胺的变化特别敏感。相反，切割活性仅对嘌呤核的7位修饰敏感。这些结果表明，腺嘌呤碱基的沃森-克里克边缘在核酶功能中很重要。我们试图测试核碱基在裂解反应化学中直接作用的可能性。在培养基中添加咪唑碱无法恢复核酶的活性，从该酶中去除了​​A756的核碱基。然而，用外源腺嘌呤碱也没有得到修复，表明可能由于碱的消融而导致的腔关闭。

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