Fersht A R, Mulvey R S, Koch G L
Biochemistry. 1975 Jan 14;14(1):13-8. doi: 10.1021/bi00672a003.
The interaction of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus with its substrates in the aminoacyl adenylation reaction has been studied by stopped-flow fluorescence. The observed changes have been assigned to their chemical and physical processes by comparison with equilibrium dialysis, pyrophosphate exchange kinetics and rapid quenching and sampling techniques to give the rate constants for ligand binding, the formation of tyrosyl adenylate, and the reverse reaction. The stoichiometry of tyrosine and ATP binding in the catalytic process has been determined directly by equilibrium dialysis and equilibrium gel filtration under pyrophosphate exchange conditions, i.e., where a steady state has been set up in which the equilibrium position favors starting materials. It is shown that the rate-determining step in the formation of tyrosyl adenylate involves 1 mole each of tyrosine and ATP. A second mole of tyrosine and ATP bind to the aminoacyl adenylate complex stabilizing the high-energy intermediate. The enzyme tyrosyl adenylate complex that is isolated by gel filtration is in a different conformational state from that in the presence of tyrosine and ATP.
利用停流荧光法研究了嗜热脂肪芽孢杆菌酪氨酰 - tRNA合成酶在氨酰腺苷酸化反应中与其底物的相互作用。通过与平衡透析、焦磷酸交换动力学以及快速淬灭和取样技术相比较，将观察到的变化归因于其化学和物理过程，从而得出配体结合、酪氨酰腺苷酸形成以及逆反应的速率常数。在焦磷酸交换条件下，即建立了有利于起始原料的平衡位置的稳态时，通过平衡透析和平衡凝胶过滤直接测定了催化过程中酪氨酸和ATP结合的化学计量。结果表明，酪氨酰腺苷酸形成过程中的速率决定步骤涉及1摩尔酪氨酸和1摩尔ATP。第二摩尔酪氨酸和ATP与氨酰腺苷酸复合物结合，稳定高能中间体。通过凝胶过滤分离得到的酶 - 酪氨酰腺苷酸复合物与存在酪氨酸和ATP时的构象状态不同。
