The homotetrameric phosphoseryl-tRNA synthetase from Methanosarcina mazei exhibits half-of-the-sites activity
The homotetrameric phosphoseryl-tRNA synthetase from Methanosarcina mazei exhibits half-of-the-sites activity
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DOI：
10.1074/jbc.m801838200
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发表时间：
2008-08-08
影响因子：
4.8
通讯作者：
Perona, John J.
中科院分区：
文献类型：
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作者：
Hauenstein, Scott I.;Hou, Ya-Ming;Perona, John J.
在产甲烷的古细菌中，半胱氨酸- trna （Cys）的合成是通过两步途径进行的，在这两步途径中，trys首先通过磷酸丝氨酸- trna合成酶（sepr）与磷酸丝氨酸氨基化。凝胶过滤和非变性质谱分析表明，该天然酶在大肠杆菌中高水平表达时以α 4四聚体的形式存在。然而，通过ATP/PPi爆发动力学监测的活性位点滴定以及荧光光谱分析的tRNA结合化学计量学表明，四聚体酶结合两个tRNA，并且四个化学等效亚基中只有两个催化磷酸丝氨酸酯的形成。因此，先前描述的由二聚体I类酪氨酸-tRNA合成酶合成1mol酪氨酸腺苷酸的半位点活性现象，也适用于这种同四聚体II类tRNA合成酶。通过ATP/PPi和氨基酰化动力学分析同源和非同源反应强烈表明，sepr能够区分非同源氨基酸谷氨酸、丝氨酸和磷苏氨酸，而不需要单独的水解编辑位点。tRNACys与sepr的结合也增强了酶区分氨基酸的能力，表明tRNA与酶的氨基酸结合位点之间存在功能连通性。
Synthesis of cysteinyl-tRNA(Cys) in methanogenic archaea proceeds by a two-step pathway in which tRNACys is first aminoacylated with phosphoserine by phosphoseryl-tRNA synthetase (SepRS). Characterization of SepRS from the mesophile Methanosarcina mazei by gel filtration and nondenaturing mass spectrometry shows that the native enzyme exists as an alpha 4 tetramer when expressed at high levels in Escherichia coli. However, active site titrations monitored by ATP/PPi burst kinetics, together with analysis of tRNA binding stoichiometry by fluorescence spectroscopy, show that the tetrameric enzyme binds two tRNAs and that only two of the four chemically equivalent subunits catalyze formation of phosphoseryl adenylate. Therefore, the phenomenon of half-of-the-sites activity, previously described for synthesis of 1 mol of tyrosyl adenylate by the dimeric class I tyrosyl-tRNA synthetase, operates as well in this homotetrameric class II tRNA synthetase. Analysis of cognate and noncognate reactions by ATP/PPi and aminoacylation kinetics strongly suggests that SepRS is able to discriminate against the noncognate amino acids glutamate, serine, and phosphothreonine without the need for a separate hydrolytic editing site. tRNACys binding to SepRS also enhances the capacity of the enzyme to discriminate among amino acids, indicating the existence of functional connectivity between the tRNA and amino acid binding sites of the enzyme.