Rath V L, Silvian L F, Beijer B, Sproat B S, Steitz T A
Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, PO Box 208114, New Haven, CT 06520-8114, USA.
Structure. 1998 Apr 15;6(4):439-49. doi: 10.1016/s0969-2126(98)00046-x.
Aminoacyl-tRNA synthetases covalently link a specific amino acid to the correct tRNA. The fidelity of this reaction is essential for accurate protein synthesis. Each synthetase has a specific molecular mechanism to distinguish the correct pair of substrates from the pool of amino acids and isologous tRNA molecules. In the case of glutaminyl-tRNA synthetase (GlnRS) the prior binding of tRNA is required for activation of glutamine by ATP. A complete understanding of amino acid specificity in GlnRS requires the determination of the structure of the synthetase with both tRNA and substrates bound.
A stable glutaminly-adenylate analog, which inhibits GlnRS with a Ki of 1.32 microM, was synthesized and cocrystallized with GlnRS and tRNA2Gln. The crystal structure of this ternary complex has been refined at 2.4 A resolution and shows the interactions made between glutamine and its binding site.
To select against glutamic acid or glutamate, both hydrogen atoms of the nitrogen of the glutamine sidechain are recognized. The hydroxyl group of Tyr211 and a water molecule are responsible for this recognition; both are obligate hydrogen-bond acceptors due to a network of interacting sidechains and water molecules. The prior binding of tRNAGln that is required for amino acid activation may result from the terminal nucleotide, A76, packing against and orienting Tyr211, which forms part of the amino acid binding site.
氨酰 - tRNA合成酶将特定氨基酸共价连接到正确的tRNA上。该反应的保真度对于准确的蛋白质合成至关重要。每种合成酶都有特定的分子机制，可从氨基酸和同源tRNA分子库中区分出正确的底物对。就谷氨酰胺 - tRNA合成酶（GlnRS）而言，tRNA的预先结合是ATP激活谷氨酰胺所必需的。要全面了解GlnRS中的氨基酸特异性，需要确定结合了tRNA和底物的合成酶结构。
合成了一种稳定的谷氨酰胺 - 腺苷酸类似物，其对GlnRS的抑制常数Ki为1.32微摩尔，将其与GlnRS和tRNA2Gln共结晶。该三元复合物的晶体结构已在2.4埃分辨率下进行了优化，显示了谷氨酰胺与其结合位点之间的相互作用。
为了区分谷氨酸或谷氨酰胺，谷氨酰胺侧链氮的两个氢原子都被识别。Tyr211的羟基和一个水分子负责这种识别；由于相互作用的侧链和水分子网络，两者都是必需的氢键受体。氨基酸激活所需的tRNAGln的预先结合可能是由于末端核苷酸A76与形成氨基酸结合位点一部分的Tyr211堆积并使其定向。
