Fournier M, Labouesse B, Labouesse J
Institut de Biochimie Cellulaire et Neurochimie, Centre National de la Recherche Scientifique, Bordeaux, France.
Eur J Biochem. 1987 Sep 15;167(3):533-40. doi: 10.1111/j.1432-1033.1987.tb13370.x.
Tryptophanyl-tRNA synthetase from beef pancreas reacts with periodate-oxidized ATP according to biphasic kinetics. A rapid phase involves two groups of the protein, presumably lysine side-chains. The slow phase corresponds to the reaction of a larger number of groups. The time-course of the partial losses of the ATP-PPi isotopic exchange and of the aminoacylation activities of the enzyme follow the labelling of the two fast-reacting groups. However, the ability of the enzyme to form a bis(tryptophanyladenylate)-enzyme complex is not lost after reaction of these two groups with the reagent. The affinity for ATP is also unaffected by this initial labelling of the protein, as seen from the Km values of this substrate in the ATP-PPi isotopic exchange reaction. These data suggest that, in this fast initial reaction, oxidized ATP reacts neither with specific ATP-binding groups of the enzyme nor with any major catalytic residue of the tryptophan-activation site. In contrast with this first step, the further slow labelling of lysine residues leads to a disappearance of the aminoacylation ability of the enzyme, while it does not further affect the ATP-PPi exchange activity. The behaviour of beef tryptophanyl-tRNA synthetase during derivatization with oxidized ATP is therefore at variance with that which has been described for the homologous E. coli enzyme.
来自牛胰腺的色氨酰 - tRNA合成酶与高碘酸盐氧化的ATP按照双相动力学反应。快速相涉及蛋白质的两组基团，推测为赖氨酸侧链。慢相对应大量基团的反应。酶的ATP - PPi同位素交换和氨酰化活性部分丧失的时间进程遵循两个快速反应基团的标记情况。然而，在这两组基团与试剂反应后，酶形成双（色氨酰腺苷酸） - 酶复合物的能力并未丧失。从ATP - PPi同位素交换反应中该底物的Km值可以看出，对ATP的亲和力也不受蛋白质这一初始标记的影响。这些数据表明，在这个快速的初始反应中，氧化的ATP既不与酶的特定ATP结合基团反应，也不与色氨酸活化位点的任何主要催化残基反应。与第一步不同，赖氨酸残基的进一步缓慢标记导致酶的氨酰化能力消失，而它不再进一步影响ATP - PPi交换活性。因此，牛色氨酰 - tRNA合成酶在用氧化ATP进行衍生化过程中的行为与已描述的同源大肠杆菌酶的行为不同。
