Identification by Mn2+ rescue of two residues essential for the proton transfer of tRNase Z catalysis.
Identification by Mn2+ rescue of two residues essential for the proton transfer of tRNase Z catalysis.
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DOI：
10.1093/nar/gkl517
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发表时间：
2006
影响因子：
14.9
通讯作者：
Nashimoto M
中科院分区：
文献类型：
--
作者：
Minagawa A;Takaku H;Ishii R;Takagi M;Yokoyama S;Nashimoto M
Thermotoga maritima tRNase Z恰好在A76残基之后切割含有74CCA76序列的前tRNA，形成成熟的3‘末端。它的晶体结构揭示了金属-αβ/βα-内酰胺酶超家族中典型的四层β三明治折叠。保守的6个组氨酸和2个天冬氨酸残基与金属离子一起形成了tRNase Z催化中心。在这里，我们研究了在催化中心含有单一氨基酸取代的tRNase Z变体，用于切割前tRNA。在有镁离子存在的情况下，几乎没有检测到每个变体的切割，尽管它与前tRNA结合。然而，令人惊讶的是，除Asp52Ala和His222Ala替换外，Mn2+离子恢复了失去的依赖于镁的活性，这两个替换几乎完全取消了该活性。这些结果为Asp-52和His-222对催化质子转移的直接贡献提供了证据。
Thermotoga maritima tRNase Z cleaves pre-tRNAs containing the 74CCA76 sequence precisely after the A76 residue to create the mature 3′ termini. Its crystal structure has revealed a four-layer αβ/βα sandwich fold that is typically found in the metallo-β-lactamase superfamily. The well-conserved six histidine and two aspartate residues together with metal ions are assumed to form the tRNase Z catalytic center. Here, we examined tRNase Z variants containing single amino acid substitutions in the catalytic center for pre-tRNA cleavage. Cleavage by each variant in the presence of Mg2+ was hardly detected, although it is bound to pre-tRNA. Surprisingly, however, Mn2+ ions restored the lost Mg2+-dependent activity with two exceptions of the Asp52Ala and His222Ala substitutions, which abolished the activity almost completely. These results provide a piece of evidence that Asp-52 and His-222 directly contribute the proton transfer for the catalysis.
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