Freist W, Cramer F
Max-Planck-Institut für experimentelle Medizin, Abteilung Chemie, Göttingen, Federal Republic of Germany.
Eur J Biochem. 1990 Jul 20;191(1):123-9. doi: 10.1111/j.1432-1033.1990.tb19101.x.
For discrimination between valine and the 19 naturally occurring noncognate amino acids, as well as between valine and 2-amino-isobutyric acid by valyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values in aminoacylation of tRNA(Val)-C-C-A. The lowest values were found for Trp, Ser, Cys, Lys, Met and Thr (D = 90-870), showing that valine is 90-870 times more frequently attached to tRNA(Val)-C-C-A than the noncognate amino acids at the same amino acid concentrations. The other amino acids exhibit D values between 1,100 and 6200. Generally, valyl-tRNA synthetase is considerably less specific than isoleucyl-tRNA synthetase, but this may be partly compensated in the cell by valine concentrations higher than those of noncognate acids. In aminoacylation of tRNA(Val)-C-C-A(3'NH2) discrimination factors D1 are in the range of 40-1260. From D1 values and AMP formation stoichiometry, pretransfer proof-reading factors pi 1 were determined: post-transfer proof-reading factors II 2 were determined from D values and AMP formation stoichiometry in acylation of tRNA(Val)-C-C-A. II 1 values (7-168) show that pretransfer proof-reading is the main correction step, post-transfer proof-reading (II 2 approximately 1-7) is less effective and in some cases negligible. Initial discrimination factors were calculated from discrimination and proof-reading factors according to a two-step binding process. These factors, due to different Gibbs free energies of binding can be related to hydrophobic interaction forces, and a hypothetical 'stopper' model of the amino-acid-binding site is discussed.
为了区分缬氨酸与19种天然存在的非同源氨基酸，以及面包酵母缬氨酰 - tRNA合成酶区分缬氨酸与2 - 氨基异丁酸，已根据tRNA(Val)-C-C-A氨酰化反应中的kcat和Km值确定了区分因子（D）。发现色氨酸、丝氨酸、半胱氨酸、赖氨酸、甲硫氨酸和苏氨酸的区分因子最低（D = 90 - 870），这表明在相同氨基酸浓度下，缬氨酸与tRNA(Val)-C-C-A结合的频率是这些非同源氨基酸的90 - 870倍。其他氨基酸的D值在1100至6200之间。一般来说，缬氨酰 - tRNA合成酶的特异性远低于异亮氨酰 - tRNA合成酶，但在细胞中，缬氨酸浓度高于非同源氨基酸的浓度可能在一定程度上弥补了这一点。在tRNA(Val)-C-C-A(3'NH2)的氨酰化反应中，区分因子D1在40 - 1260范围内。根据D1值和AMP形成化学计量，确定了转移前校对因子π1：通过tRNA(Val)-C-C-A酰化反应中的D值和AMP形成化学计量确定了转移后校对因子II 2。II 1值（7 - 168）表明转移前校对是主要的校正步骤，转移后校对（II 2约为1 - 7）效果较差，在某些情况下可忽略不计。根据两步结合过程，从区分和校对因子计算出初始区分因子。由于结合的吉布斯自由能不同，这些因子可能与疏水相互作用力有关，并讨论了氨基酸结合位点的假设“塞子”模型。
