Bartmann P, Hanke T, Holler E
J Biol Chem. 1975 Oct 10;250(19):7668-74.
The existence of two active siter per molecule of L-phenylalanine:tRNA ligase from Escherichia coli K(-10) has been demonstrated by isolation of the E-aminoacyl adenylate and tel filtration and the nitrocellulose filter assay at pH 5.0 revealed the same stoichiometry for the E-tRNAPhe comples as protection against degradation by snake venom phosphodiesterase and equilibrium gel filtration at pH 7.5. Using a fluorescence titration technique, it was found that the dissociation constant for ligase-tRNAPhe complex is decreased 20-fold when the hydrogen ion concentration is changed from pH 6.0 to pH 5.0. The existence of two active sites binding the aminoacyl adenylate intermediate was demonstrated by gel filtration and retention on DEAE-cellulose filters. "Burst" experiments indicated that two sites were involved in a rapid ATP consumption at conditions of catalytic amino acid activation. Furthermore, it was observed that the activated amino acid could be transferred from both sites to cognate tRNA.
通过分离E-氨酰腺苷酸以及在pH 5.0条件下进行凝胶过滤和硝酸纤维素滤膜分析，已证明来自大肠杆菌K（-10）的L-苯丙氨酸：tRNA连接酶每个分子存在两个活性位点。该分析显示，E-tRNAPhe复合物的化学计量与在pH 7.5条件下防止蛇毒磷酸二酯酶降解和平衡凝胶过滤的情况相同。使用荧光滴定技术发现，当氢离子浓度从pH 6.0变为pH 5.0时，连接酶-tRNAPhe复合物的解离常数降低了20倍。通过凝胶过滤和在DEAE-纤维素滤膜上的保留，证明了存在两个结合氨酰腺苷酸中间体的活性位点。“爆发”实验表明，在催化氨基酸活化的条件下，两个位点都参与了快速的ATP消耗。此外，观察到活化的氨基酸可以从两个位点转移到同源tRNA上。
