Demonstration of two reaction pathways for the aminoacylation of tRNA. Application of the pulsed quenched flow technique.
Demonstration of two reaction pathways for the aminoacylation of tRNA. Application of the pulsed quenched flow technique.
复制标题
tRNA 氨酰化的两种反应途径的演示。
DOI：
10.1021/bi00686a010
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发表时间：
1975
期刊：
影响因子：
2.9
通讯作者：
R. Jakes
中科院分区：
文献类型：
--
作者：
A. Fersht;R. Jakes
描述了一种快速混合和骤冷装置，其在150毫秒至几分钟的范围内以及常规设备的5-150毫秒的通常时间尺度内有效地操作。这已被用于测量在酶的第一次周转期间由大肠杆菌的酪氨酰-tRNA合成酶酰化tRNATyr的初始速率，以及氨基酸活化和转移到tRNA的部分反应的速率常数。结果表明，在饱和浓度下，反应按三元络合物机理进行。速率决定步骤是氨酰化过程或其之前的一个步骤。在低浓度的tRNA下，反应通过形成酪氨酰腺苷酸的逐步过程进行，随后是tRNA的酰化。这些部分反应的速率常数比三元络合物反应的速率常数快。但tRNA的优先结合大大降低了酪氨酰腺苷酸的形成速率。这两种途径在生理浓度下可能都很重要。来自酪氨酰腺苷酸复合物的88%的酪氨酸被转移到tRNA。添加酪氨酸和ATP的存在将其降低至78%。然而，向酶、酪氨酸和饱和浓度的tRNA（即，三元复合物条件）导致至少0.97 mol tRNA被酰化/mol ATP水解。捕获实验表明，未转移到tRNA的12%的腺苷酸化在酶上水解，而不是排入溶液。
A rapid mixing and quenching device is described which operates efficiently in the range of 150 msec to several minutes as well as the usual time scale of 5-150 msec of the conventional apparatus. This has been used to measure the initial rate of acylation of tRNATyr by the tyrosyl-tRNA synthetase of Escherichia coli during the first turnover of the enzyme, and also the rate constants of the partial reactions of amino acid activation and transfer to the tRNA. It is shown that at saturating conenctration of tRNA the reaction proceeds by a ternary complex mechanism. The rate-determining step is either the aminoacyltion process or a step preceding it. At low concentrations of tRNA the reaction proceeds by the stepwise process of formation of tyrosyl adenylate followed by acylation of the tRNA. The rate constants for these partial reactions are faster than that for the ternary complex reaction. But the prior binding of tRNA greatly decreases the rate of tyrosyl adenylate formation. Both pathways are probably important at physiological concentrations. 88% of the tyrosine from the tyrosyl adenylate complex is transferred to tRNA. The presence of added tyrosine and ATP reduces this to 78%. However, the addition of aliquots of ATP to a mixture of enzyme, tyrosine, and a saturating concentration of tRNA (i.e., ternary complex conditions) leads to at least 0.97 mol of tRNA being acylated/mol of ATP hydrolyzed. Trapping experiments show that the 12% of adenylated that is not transferred to tRNA is hydrolyzed on the enzyme rather than expelled into solution.