Histidyl-tRNA synthetase urzymes: Class I and II aminoacyl tRNA synthetase urzymes have comparable catalytic activities for cognate amino acid activation.
Histidyl-tRNA synthetase urzymes: Class I and II aminoacyl tRNA synthetase urzymes have comparable catalytic activities for cognate amino acid activation.
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DOI：
10.1074/jbc.m110.198929
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发表时间：
2011-03-25
期刊：
影响因子：
--
通讯作者：
Carter CW Jr
中科院分区：
文献类型：
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作者：
Li L;Weinreb V;Francklyn C;Carter CW Jr
构建了大肠杆菌II类组氨酰-tRNA合成酶的四个最小（119 - 145个残基）活性位点片段，表达为麦芽糖结合蛋白融合体，并使用32 PPi交换测定法测定融合蛋白和TEV切割后的组氨酸活化。它们都含有保守的基序1和2。两个含有基序1的N-末端延伸，两个含有基序3。五个实验结果有力地证明了所观察到的催化活性的真实性：（i）活性位点滴定实验显示高的（ii）通过TEV切割融合蛋白释放隐藏活性，（iii）与活性位点突变相关的活性降低，（iv）将增加的催化活性定量归因于基序3的内在效应，N-末端延伸和它们的协同效应，以及（v）显著改变ATP和组氨酸底物的Km值。因此，似乎插入结构域和基序3都不是最早的II类氨酰-tRNA合成酶的催化活性所必需的。所有四个切割的构建体的平均速率增强是估计的未催化速率的109倍。如对于双甘氨酰-tRNA合成酶（TrpRS）Urzyme所观察到的，这些片段紧密结合ATP，但对同源氨基酸具有降低的亲和力。因此，这些片段可能代表Urzyme（Ur =原始的、原始的、最早的+酶），其大小和催化活性相当，并且由与编码先前描述的I类TrpRS Urzyme的序列反义的序列编码。它们的催化活性提供了非常早期的进化事件的实验重演的指标。
Four minimal (119 - 145 residue) active site fragments of Escherichia coli Class II histidyl-tRNA synthetase were constructed, expressed as maltose-binding protein fusions, and assayed for histidine activation as fusion proteins and after TEV cleavage, using the 32PPi exchange assay. All contain conserved Motifs 1 and 2. Two contain an N-terminal extension of Motif 1 and two contain Motif 3. Five experimental results argue strongly for the authenticity of the observed catalytic activities: (i) active site titration experiments showing high (∼0.1–0.55) fractions of active molecules, (ii) release of cryptic activity by TEV cleavage of the fusion proteins, (iii) reduced activity associated with an active site mutation, (iv) quantitative attribution of increased catalytic activity to the intrinsic effects of Motif 3, the N-terminal extension and their synergistic effect, and (v) significantly altered Km values for both ATP and histidine substrates. It is therefore plausible that neither the insertion domain nor Motif 3 were essential for catalytic activity in the earliest Class II aminoacyl-tRNA synthetases. The mean rate enhancement of all four cleaved constructs is ∼109 times that of the estimated uncatalyzed rate. As observed for the tryptophanyl-tRNA synthetase (TrpRS) Urzyme, these fragments bind ATP tightly but have reduced affinity for cognate amino acids. These fragments thus likely represent Urzymes (Ur = primitive, original, earliest + enzyme) comparable in size and catalytic activity and coded by sequences proposed to be antisense to that coding the previously described Class I TrpRS Urzyme. Their catalytic activities provide metrics for experimental recapitulation of very early evolutionary events.