Freist W, Sternbach H
Max-Planck-Institut für Experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
Eur J Biochem. 1989 Jan 2;178(3):595-602. doi: 10.1111/j.1432-1033.1989.tb14487.x.
Specificity with regard to amino acids in aminoacylation of tRNA(Ile)-C-C-3'dA by isoleucyl-tRNA synthetase is characterized by discrimination factors (D2) which are calculated from kcat and Km values. The lowest values are observed for Cys, Val, His, and Trp (D2 = 180-1700), indicating that at same amino acid concentrations isoleucine is 180-1700 times more attached to tRNA(Ile)-C-C-3'dA. The highest values are observed for Gly, Ala, Ser, Pro, Gln, Leu, Glu, and Phe (D2 = 10,000-30,000). D2 values of the other amino acids are in the range of 2000-10,000. Recognition of most amino acids is achieved in a four-step process. Two initial discrimination steps are due to different hydrophobic interactions with the binding pockets; two proof-reading steps occur on the pre- and the post-transfer stage. For nine amino acids (Ser, Asp, Asn, Val, Leu, His, Phe, Lys, Trp) post-transfer proof-reading is negligible. As a special case in discrimination of valine, one initial discrimination step and the post-transfer proof-reading step are lacking. The role of the terminal hydroxyl groups of the tRNA for post-transfer proof-reading is assigned to a simple neighbouring group effect. No preference for the 2' or 3' position in proof-reading can be postulated.
异亮氨酰 - tRNA合成酶对tRNA（Ile）-C-C-3'dA进行氨酰化时，针对氨基酸的特异性由判别因子（D2）来表征，该因子根据kcat和Km值计算得出。半胱氨酸、缬氨酸、组氨酸和色氨酸的D2值最低（D2 = 180 - 1700），这表明在相同氨基酸浓度下，异亮氨酸与tRNA（Ile）-C-C-3'dA的结合能力是其他氨基酸的180 - 1700倍。甘氨酸、丙氨酸、丝氨酸、脯氨酸、谷氨酰胺、亮氨酸、谷氨酸和苯丙氨酸的D2值最高（D2 = 10,000 - 30,000）。其他氨基酸的D2值在2000 - 10,000范围内。大多数氨基酸的识别通过四个步骤实现。最初的两个判别步骤是由于与结合口袋的疏水相互作用不同；两个校对步骤分别发生在转移前和转移后阶段。对于九种氨基酸（丝氨酸、天冬氨酸、天冬酰胺、缬氨酸、亮氨酸、组氨酸、苯丙氨酸、赖氨酸、色氨酸），转移后校对可忽略不计。作为缬氨酸判别中的一个特殊情况，缺少一个初始判别步骤和转移后校对步骤。tRNA末端羟基在转移后校对中的作用归因于一种简单的邻基效应。在校对过程中，无法假定对2'或3'位置有偏好。
