Bullock Timothy L, Uter Nathan, Nissan T Amar, Perona John J
Department of Chemistry and Biochemistry, and Interdepartmental Program in Biomolecular Science and Engineering, University of California at Santa Barbara, Santa Barbara, CA 93106-9510, USA.
J Mol Biol. 2003 Apr 25;328(2):395-408. doi: 10.1016/s0022-2836(03)00305-x.
The 2.5 A crystal structure of Escherichia coli glutaminyl-tRNA synthetase in a quaternary complex with tRNA(Gln), an ATP analog and glutamate reveals that the non-cognate amino acid adopts a distinct binding mode within the active site cleft. In contrast to the binding of cognate glutamine, one oxygen of the charged glutamate carboxylate group makes a direct ion-pair interaction with the strictly conserved Arg30 residue located in the first half of the dinucleotide fold domain. The nucleophilic alpha-carboxylate moiety of glutamate is mispositioned with respect to both the ATP alpha-phosphate and terminal tRNA ribose groups, suggesting that a component of amino acid discrimination resides at the catalytic step of the reaction. Further, the other side-chain carboxylate oxygen of glutamate is found in a position identical to that previously proposed to be occupied by the NH(2) group of the cognate glutamine substrate. At this position, the glutamate oxygen accepts hydrogen bonds from the hydroxyl moiety of Tyr211 and a water molecule. These findings demonstrate that amino acid specificity by GlnRS cannot arise from hydrogen bonds donated by the cognate glutamine amide to these same moieties, as previously suggested. Instead, Arg30 functions as a negative determinant to drive binding of non-cognate glutamate into a non-productive orientation. The poorly differentiated cognate amino acid-binding site in GlnRS may be a consequence of the late emergence of this enzyme from the eukaryotic lineage of glutamyl-tRNA synthetases.
大肠杆菌谷氨酰胺-tRNA合成酶与tRNA(Gln)、一种ATP类似物和谷氨酸形成的四元复合物的2.5埃晶体结构表明，非同源氨基酸在活性位点裂隙内采用独特的结合模式。与同源谷氨酰胺的结合不同，带电荷的谷氨酸羧酸盐基团的一个氧原子与位于二核苷酸折叠结构域前半部分的严格保守的Arg30残基形成直接的离子对相互作用。谷氨酸的亲核α-羧酸盐部分相对于ATP的α-磷酸基团和末端tRNA核糖基团位置错误，这表明氨基酸识别的一个组成部分存在于反应的催化步骤中。此外，发现谷氨酸的另一个侧链羧酸盐氧原子所处位置与先前提出的同源谷氨酰胺底物的NH(2)基团所占据的位置相同。在此位置，谷氨酸的氧原子接受来自Tyr211的羟基部分和一个水分子的氢键。这些发现表明，谷氨酰胺-tRNA合成酶的氨基酸特异性并非如先前所认为的那样源于同源谷氨酰胺酰胺向这些相同部分提供的氢键。相反，Arg30作为一个负向决定因素，将非同源谷氨酸的结合驱动到非生产性方向。谷氨酰胺-tRNA合成酶中同源氨基酸结合位点的差异不大可能是该酶从真核生物谷氨酰胺-tRNA合成酶谱系中较晚出现的结果。
