Konno Michiko, Sumida Tomomi, Uchikawa Emiko, Mori Yukie, Yanagisawa Tatsuo, Sekine Shun-ichi, Yokoyama Shigeyuki
Department of Chemistry and Biochemistry, Graduate School of Humanities and Sciences, Ochanomizu University, Tokyo, Japan.
FEBS J. 2009 Sep;276(17):4763-79. doi: 10.1111/j.1742-4658.2009.07178.x. Epub 2009 Jul 27.
The ATP-pyrophosphate exchange reaction catalyzed by Arg-tRNA, Gln-tRNA and Glu-tRNA synthetases requires the assistance of the cognate tRNA. tRNA also assists Arg-tRNA synthetase in catalyzing the pyrophosphorolysis of synthetic Arg-AMP at low pH. The mechanism by which the 3'-end A76, and in particular its hydroxyl group, of the cognate tRNA is involved with the exchange reaction catalyzed by those enzymes has yet to be established. We determined a crystal structure of a complex of Arg-tRNA synthetase from Pyrococcus horikoshii, tRNA(Arg)(CCU) and an ATP analog with Rfactor = 0.213 (Rfree = 0.253) at 2.0 A resolution. On the basis of newly obtained structural information about the position of ATP bound on the enzyme, we constructed a structural model for a mechanism in which the formation of a hydrogen bond between the 2'-OH group of A76 of tRNA and the carboxyl group of Arg induces both formation of Arg-AMP (Arg + ATP --> Arg-AMP + pyrophosphate) and pyrophosphorolysis of Arg-AMP (Arg-AMP + pyrophosphate --> Arg + ATP) at low pH. Furthermore, we obtained a structural model of the molecular mechanism for the Arg-tRNA synthetase-catalyzed deacylation of Arg-tRNA (Arg-tRNA + AMP --> Arg-AMP + tRNA at high pH), in which the deacylation of aminoacyl-tRNA bound on Arg-tRNA synthetase and Glu-tRNA synthetase is catalyzed by a quite similar mechanism, whereby the proton-donating group (-NH-C+(NH2)2 or -COOH) of Arg and Glu assists the aminoacyl transfer from the 2'-OH group of tRNA to the phosphate group of AMP at high pH.
精氨酸 - tRNA合成酶、谷氨酰胺 - tRNA合成酶和谷氨酸 - tRNA合成酶催化的ATP - 焦磷酸交换反应需要相应tRNA的协助。tRNA还能在低pH条件下协助精氨酸 - tRNA合成酶催化合成的精氨酰 - AMP的焦磷酸解反应。相应tRNA的3'-末端A76，特别是其羟基，参与这些酶催化的交换反应的机制尚未明确。我们测定了来自嗜热栖热菌的精氨酸 - tRNA合成酶、tRNA(Arg)(CCU)和一种ATP类似物复合物的晶体结构，分辨率为2.0 Å，R因子 = 0.213（R自由 = 0.253）。基于新获得的关于ATP在酶上结合位置的结构信息，我们构建了一个结构模型，该模型表明在低pH条件下，tRNA的A76的2'-OH基团与精氨酸的羧基之间形成氢键会诱导精氨酰 - AMP的形成（精氨酸 + ATP → 精氨酰 - AMP + 焦磷酸）以及精氨酰 - AMP的焦磷酸解（精氨酰 - AMP + 焦磷酸 → 精氨酸 + ATP）。此外，我们还获得了精氨酸 - tRNA合成酶催化精氨酸 - tRNA脱酰基反应（在高pH条件下，精氨酸 - tRNA + AMP → 精氨酰 - AMP + tRNA）的分子机制的结构模型，其中结合在精氨酸 - tRNA合成酶和谷氨酸 - tRNA合成酶上的氨酰 - tRNA的脱酰基反应是由非常相似的机制催化的，即精氨酸和谷氨酸的质子供体基团（-NH - C+(NH2)2或 -COOH）在高pH条件下协助氨酰基从tRNA 的2'-OH基团转移到AMP的磷酸基团上。
