Acchione Mauro, Guillemette Joseph G, Twine Susan M, Hogue Christopher W V, Rajendran Bahe, Szabo Arthur G
Wilfrid Laurier University, Waterloo, Canada N2L 3C5.
Biochemistry. 2003 Dec 23;42(50):14994-5002. doi: 10.1021/bi0347454.
The symmetrical dimer structure of tryptophanyl-tRNA synthetase is similar to that of tyrosyl-tRNA synthetase whose binding behavior and structural details have been elucidated in detail. The structure of both subunits after forming the intermediate tryptophanyl-AMP has important implications for the binding of the cognate tRNA(Trp). Single tryptophan mutants of Bacillus stearothermophilus tryptophanyl-tRNA synthetase have been constructed and expressed and used to probe structural changes in different domains of the enzyme in both subunits. Substrate titrations using the Trp analogues 4-fluorotryptophan and 7-azatryptophan in the presence of ATP to form the corresponding aminoacyl-adenylate reveal significant structural changes occurring throughout the active subunit in regions not confined to the active site. Changes in environment around the specific Trp residues were monitored using UV absorbance and steady-state fluorescence measurements. When titrated with 4-fluorotryptophan, both Trp 91 and Trp 290 fluorescence is quenched (49 and 22%, respectively) when one subunit has formed Trp-AMP. The fluorescence of Trp 48 is enhanced 19%. No further change in signal was observed after a 1:1 dimer/L-4FW-AMP complex ratio had been established. Using an anion-exchange filter binding assay with radiolabeled l-Trp as a substrate, binding to only one subunit was observed under nonsaturating conditions. This agrees with the results of the assay using 7-azatryptophan as a substrate. The observed changes extend to the unfilled subunit where a similar structure is believed to form after one subunit has formed tryptophan-AMP. Movement in the regions of the enzyme containing Trp 290 and Trp 91 suggests a mechanism for cross-subunit communication involving the helical backbone and dimer interface containing these two residues.
色氨酰 - tRNA合成酶的对称二聚体结构与酪氨酰 - tRNA合成酶的结构相似，后者的结合行为和结构细节已得到详细阐明。形成中间产物色氨酰 - AMP后，两个亚基的结构对同源tRNA（Trp）的结合具有重要意义。已构建并表达了嗜热栖热放线菌色氨酰 - tRNA合成酶的单色氨酸突变体，并用于探测该酶两个亚基不同结构域的结构变化。在ATP存在下，使用色氨酸类似物4 - 氟色氨酸和7 - 氮杂色氨酸进行底物滴定以形成相应的氨酰 - 腺苷酸，结果显示在整个活性亚基中，不仅活性位点所在区域，其他区域也发生了显著的结构变化。利用紫外吸收和稳态荧光测量监测特定色氨酸残基周围环境的变化。用4 - 氟色氨酸滴定，当一个亚基形成色氨酰 - AMP时，Trp 91和Trp 290的荧光均被淬灭（分别为49%和22%）。Trp 48的荧光增强了19%。在建立了1:1的二聚体/L - 4FW - AMP复合物比例后，未观察到信号进一步变化。使用以放射性标记的L - Trp为底物的阴离子交换滤膜结合测定法，在非饱和条件下仅观察到与一个亚基的结合。这与使用7 - 氮杂色氨酸作为底物的测定结果一致。观察到的变化延伸至未填充的亚基，据信在一个亚基形成色氨酰 - AMP后，该亚基会形成类似的结构。含有Trp 290和Trp 91的酶区域的移动表明存在一种涉及包含这两个残基的螺旋主链和二聚体界面的亚基间通讯机制。
