Fersht A R, Ashford J S, Bruton C J, Jakes R, Koch G L, Hartley B S
Biochemistry. 1975 Jan 14;14(1):1-4. doi: 10.1021/bi00672a001.
A simple, rapid, and economical procedure is described for the determination of the number of catalytically competent active sites on aminoacyl-tRNA synthetases based on the stoichiometry of aminoacyl adenylate formation. On mixing tRNA synthetase, cognate amino acid, (gamma-32P)ATP, and inorganic pyrophosphatase under suitable conditions there is an initial rapid stoichiometric "burst" (rate constant k1) of depletion of ATP as enzyme bound aminoacyl adenylate is formed. There is then an initially linear decrease in ATP concentration as the complex hydrolyzes (with rate constant k2) releasing enzyme to form further adenylate. Provided k2 less than k1 the initial burst gives the stoichiometry of aminoacyl adenylate formation. Complexes which are too unstable to be isolated by the usual gel or nitrocellulose disk filtration procedure may be assayed in this way. This technique has been applied to five highly purified aminoacyl-tRNA synthetases. The tyrosyl-tRNA synthetase from Bacillus stearothermophilus is shown to bind only one aminoacyl adenylate per dimer.
描述了一种基于氨酰腺苷酸形成的化学计量来测定氨酰 - tRNA合成酶上催化活性位点数量的简单、快速且经济的方法。在合适的条件下，将tRNA合成酶、同源氨基酸、（γ - 32P）ATP和无机焦磷酸酶混合，随着酶结合的氨酰腺苷酸形成，ATP会有一个初始快速的化学计量“爆发”（速率常数k1）而消耗。然后，随着复合物水解（速率常数k2）释放酶以形成更多的腺苷酸，ATP浓度会有一个初始线性下降。若k2小于k1，初始爆发给出氨酰腺苷酸形成的化学计量。对于那些稳定性太差而无法通过常规凝胶或硝酸纤维素圆盘过滤程序分离的复合物，可以用这种方法进行测定。该技术已应用于五种高度纯化的氨酰 - tRNA合成酶。结果表明，嗜热脂肪芽孢杆菌的酪氨酰 - tRNA合成酶每个二聚体仅结合一个氨酰腺苷酸。
