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首页> 外文期刊>Biochemistry >Methanococcus jannaschii Prolyl-Cysteinyl-tRNA Synthetase Possesses Overlapping Amino Acid Binding Sites
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Methanococcus jannaschii Prolyl-Cysteinyl-tRNA Synthetase Possesses Overlapping Amino Acid Binding Sites

机译：詹氏甲烷球菌Prolyl-半胱氨酰-tRNA合成酶具有重叠的氨基酸结合位点

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摘要

The protein translation apparatus of Methanococcus jannaschii possesses the unusual enzyme prolyl-cysteinyl-tRNA synthetase (ProCysRS), a single enzyme that attaches two different amino acids, proline and cysteine, to their cognate tRNA species. Measurement of the ATP-PPj exchange reaction revealed that amino acid activation, the first reaction step, differs for the two amino acids. While Pro- AMP can be formed in the absence of tRNA, Cys-AMP synthesis is tRNA-dependent. Studies with purified tRNAs indicate that tRNA Cys promotes cysteine activation. The kcat values of wild-type ProCysRS for tRNA prolylation (0.09 S-I) and cysteinylation (0.02 S-I) demonstrate that both aminoacyl-tRNAs are synthesized with comparable rates, the cysteinyl-tRNA synthetase activity being only 4.5-fold lower than prolyl-tRNA synthetase activity. Kinetic analysis of ProCysRS mutant enzymes, generated by site-directed mutagenesis, shows glutamate at position 103 to be critical for proline binding, and proline at position 100 to be involved in cysteine binding. The proximity in ProCysRS of amino acid residues affecting binding of either cysteine or proline strongly suggests that structural elements of the two amino acid binding sites overlap.
机译：詹氏甲烷球菌的蛋白质翻译装置具有不同寻常的酶脯氨酰-半胱氨酰-tRNA合成酶（ProCysRS），该酶将两个不同的氨基酸脯氨酸和半胱氨酸连接到它们的同源tRNA物种。 ATP-PPj交换反应的测量结果表明，氨基酸激活（第一步反应）对于这两个氨基酸有所不同。虽然Pro-AMP可以在不存在tRNA的情况下形成，但Cys-AMP的合成是tRNA依赖性的。对纯化的tRNA的研究表明，tRNA Cys促进半胱氨酸激活。野生型ProCysRS的tRNA水解（0.09 SI）和半胱氨酰化（0.02 SI）的kcat值表明，两种氨酰基-tRNA的合成率均相当，半胱氨酰-tRNA合成酶的活性仅比脯氨酰-tRNA合成酶低4.5倍活动。通过定点诱变产生的ProCysRS突变酶的动力学分析显示，位置103的谷氨酸对脯氨酸结合至关重要，而位置100的脯氨酸参与半胱氨酸结合。影响半胱氨酸或脯氨酸结合的氨基酸残基在ProCysRS中的接近性强烈表明两个氨基酸结合位点的结构元件重叠。

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