Plohl M, Kućan Z
Rudjer Bosković Institute, Zagreb, Yugoslavia.
Biochimie. 1988 May;70(5):637-44. doi: 10.1016/0300-9084(88)90247-7.
Stimulatory effects of Mg2+ and spermine on the kinetics of the aminoacylation of tRNA(Tyr) were examined using purified yeast tRNA(Tyr) and tyrosyl-tRNA synthetase. The apparent Km for tRNA(Tyr) was the lowest at Mg2+ concentrations between 2 and 5 mM and was not influenced by spermine. In the absence of spermine, the apparent Vmax was the highest at Mg2+ concentrations of 5 mM or higher, whereas the presence of spermine strongly stimulated the reaction at lower Mg2+ concentrations. Spermine alone could not substitute for Mg2+, nor was it able, at any Mg2+ concentration, to increase the reaction rate above the level reached at high concentrations of Mg2+ alone. Calculations of the concentration of Mg3.tRNA(Tyr) complex as a function of initial Mg2+ concentration, using the binding constants derived from physical measurements, allow the conclusion that spermine exerts its stimulatory activity by creating strong binding sites for Mg2+; this would enable the tRNA to assume the conformation required for optimal aminoacylation. The conformational requirement for the first tRNA: synthetase encounter is obviously less stringent, since the apparent Km for tRNA(Tyr) is not influenced by spermine.
使用纯化的酵母tRNA（Tyr）和酪氨酰-tRNA合成酶，研究了Mg2+和精胺对tRNA（Tyr）氨酰化动力学的刺激作用。tRNA（Tyr）的表观Km在Mg2+浓度为2至5 mM之间时最低，且不受精胺影响。在没有精胺的情况下，表观Vmax在Mg2+浓度为5 mM或更高时最高，而精胺的存在在较低Mg2+浓度下强烈刺激反应。单独的精胺不能替代Mg2+，在任何Mg2+浓度下，它也不能使反应速率高于仅在高浓度Mg2+时达到的水平。利用从物理测量中得出的结合常数，计算Mg3.tRNA（Tyr）复合物浓度作为初始Mg2+浓度的函数，得出结论：精胺通过为Mg2+创造强结合位点发挥其刺激活性；这将使tRNA能够呈现出最佳氨酰化所需的构象。由于tRNA（Tyr）的表观Km不受精胺影响，因此第一次tRNA与合成酶相遇时的构象要求显然不那么严格。
