Freist W, Sternbach H
Abteilung Chemie, Max-Planck-Institut für Experimentelle Medizin, Göttingen, Federal Republic of Germany.
Eur J Biochem. 1988 Nov 1;177(2):425-33. doi: 10.1111/j.1432-1033.1988.tb14392.x.
The order of substrate addition to tyrosyl-tRNA synthetase from baker's yeast was investigated by bisubstrate kinetics, product inhibition and inhibition by dead-end inhibitors. The kinetic patterns are consistent with a random bi-uni uni-bi ping-pong mechanism. Substrate specificity with regard to ATP analogs shows that the hydroxyl groups of the ribose moiety and the amino group in position 6 of the base are essential for recognition of ATP as substrate. Specificity with regard to amino acids is characterized by discrimination factors D which are calculated from kcat and Km values obtained in aminoacylation of tRNATyr-C-C-A. The lowest values are observed for Cys, Phe, Trp (D = 28,000-40,000), showing that, at the same amino acid concentrations, tyrosine is 28,000-40,000 times more often attached to tRNATyr-C-C-A than the noncognate amino acids. With Gly, Ala and Ser no misacylation could be detected (D greater than 500,000); D values of the other amino acids are in the range of 100,000-500,000. Lower specificity is observed in aminoacylation of the modified substrate tRNATyr-C-C-A(3'NH2) (D1 = 500-55,000). From kinetic constants and AMP-formation stoichiometry observed in aminoacylation of this tRNA species, as well as in acylating tRNATyr-C-C-A hydrolytic proof-reading factors could be calculated for a pretransfer (II 1) and a post-transfer (II 2) proof-reading step. The observed values of II 1 = 12-280 show that pretransfer proof-reading is the main correction step whereas post-transfer proof-reading is marginal for most amino acids (II 2 = 1-2). Initial discrimination factors caused by differences in Gibbs free energies of binding between tyrosine and noncognate amino acids are calculated from discrimination and proof-reading factors. Assuming a two-step binding process, two factors (I1 and I2) are determined which can be related to hydrophobic interaction forces. The tyrosine side chain is bound by hydrophobic forces and hydrogen bonds formed by its hydroxyl group. A hypothetical model of the amino acid binding site is discussed and compared with results of X-ray analysis of the enzyme from Bacillus stearothermophilus.
通过双底物动力学、产物抑制和终产物抑制剂抑制作用，研究了面包酵母酪氨酰 - tRNA合成酶添加底物的顺序。动力学模式与随机双单 单双乒乓机制一致。关于ATP类似物的底物特异性表明，核糖部分的羟基和碱基第6位的氨基对于将ATP识别为底物至关重要。关于氨基酸的特异性由判别因子D表征，该因子根据在tRNATyr - C - C - A氨酰化过程中获得的kcat和Km值计算得出。半胱氨酸、苯丙氨酸、色氨酸的D值最低（D = 28,000 - 40,000），这表明在相同氨基酸浓度下，酪氨酸与tRNATyr - C - C - A结合的频率比非同源氨基酸高28,000 - 40,000倍。对于甘氨酸、丙氨酸和丝氨酸，未检测到错误氨酰化（D大于500,000）；其他氨基酸的D值在100,000 - 500,000范围内。在修饰底物tRNATyr - C - C - A(3'NH2)的氨酰化过程中观察到较低的特异性（D1 = 500 - 55,000）。根据在该tRNA物种氨酰化过程中观察到的动力学常数和AMP形成化学计量，以及在酰化tRNATyr - C - C - A过程中，可以计算出转移前（II 1）和转移后（II 2）校对步骤的水解校对因子。观察到的II 1 = 12 - 280值表明转移前校对是主要的校正步骤，而转移后校对对于大多数氨基酸来说是微不足道的（II 2 = 1 - 2）。由酪氨酸和非同源氨基酸之间结合吉布斯自由能差异引起的初始判别因子根据判别和校对因子计算得出。假设一个两步结合过程，确定了两个因子（I1和I），它们可以与疏水相互作用力相关。酪氨酸侧链通过疏水作用力和其羟基形成的氢键结合。讨论了氨基酸结合位点的假设模型，并与嗜热脂肪芽孢杆菌酶的X射线分析结果进行了比较。
