Glutamate versus glutamine exchange swaps substrate selectivity in tRNA-guanine transglycosylase: insight into the regulation of substrate selectivity by kinetic and crystallographic studies.
Glutamate versus glutamine exchange swaps substrate selectivity in tRNA-guanine transglycosylase: insight into the regulation of substrate selectivity by kinetic and crystallographic studies.
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谷氨酸与谷氨酰胺交换交换 tRNA-鸟嘌呤转糖基酶中的底物选择性：通过动力学和晶体学研究深入了解底物选择性的调节。
DOI：
10.1016/j.jmb.2007.09.062
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发表时间：
2007
影响因子：
5.6
通讯作者：
Reuter,Klaus
中科院分区：
文献类型：
--
作者：
Tidten,Naomi;Stengl,Bernhard;Heine,Andreas;Garcia,GeorgeA;Klebe,Gerhard;Reuter,Klaus
细菌tRNA-鸟嘌呤转糖基酶（Tgt）催化特定tRNA的摆动位置上的鸟嘌呤被修饰的碱基preQ 1交换。然而，在体外，这种酶也能够将直接生物合成的前体preQ 0插入到这些tRNA中。这种底物混杂是基于活性位点中的肽开关，由一般的酸/碱Glu 235门控。这个开关改变了结合口袋的性质，允许鸟嘌呤或preQ 1的住宿。然而，识别鸟嘌呤的肽构象异构体也能够结合preQ 0。为了研究选择性调节，记录运动发酵单胞菌Tgt的动力学数据。他们表明，对实际底物preQ 1优于preQ 0的选择性不是通过亲和力的差异而是通过更高的周转率来实现的。此外，构建了Tgt（Glu 235 Gln）变体。该突变旨在使肽开关稳定在有利于鸟嘌呤和preQ 0结合的构象中。突变的酶的动力学特性表明，Glu 235 Gln交换，相对于所有的底物碱基，没有显着的影响KCAT。相反，KM（preQ 1）急剧增加，而KM（preQ 0）似乎减少。因此，考虑到kcat/Km作为催化效率的指标，Tgt（Glu 235 Gln）的有利于preQ 1的选择性被消除或甚至被逆转而有利于preQ 0。突变的酶的晶体结构证实，突变强烈有利于鸟嘌呤和preQ 0的住宿所需的结合口袋构象。然而，实现这一点的方式与基于野生型Tgt的晶体结构预测的方式显著不同。
Bacterial tRNA-guanine transglycosylase (Tgt) catalyses the exchange of guanine in the wobble position of particular tRNAs by the modified base preQ1. In vitro, however, the enzyme is also able to insert the immediate biosynthetic precursor, preQ0, into those tRNAs. This substrate promiscuity is based on a peptide switch in the active site, gated by the general acid/base Glu235. The switch alters the properties of the binding pocket to allow either the accommodation of guanine or preQ1. The peptide conformer recognising guanine, however, is also able to bind preQ0. To investigate selectivity regulation, kinetic data for Zymomonas mobilis Tgt were recorded. They show that selectivity in favour of the actual substrate preQ1over preQ0is not achieved by a difference in affinity but via a higher turnover rate. Moreover, a Tgt(Glu235Gln) variant was constructed. The mutation was intended to stabilise the peptide switch in the conformation favouring guanine and preQ0binding. Kinetic characterisation of the mutated enzyme revealed that the Glu235Gln exchange has, with respect to all substrate bases, no significant influence on kcat. In contrast, KM(preQ1) is drastically increased, while KM(preQ0) seems to be decreased. Hence, regarding kcat/KMas an indicator for catalytic efficiency, selectivity of Tgt in favour of preQ1is abolished or even inverted in favour of preQ0for Tgt(Glu235Gln). Crystal structures of the mutated enzyme confirm that the mutation strongly favours the binding pocket conformation required for the accommodation of guanine and preQ0. The way this is achieved, however, significantly differs from that predicted based on crystal structures of wild-type Tgt.