Théobald A, Kern D, Giegé R
Laboratoire de Biochimie, Centre National de la Recherche Scientifique, Strasbourg, France.
Biochimie. 1988 Feb;70(2):205-13. doi: 10.1016/0300-9084(88)90062-4.
Essential lysine residues were sought in the catalytic site of baker's yeast aspartyl-tRNA synthetase (an alpha 2 dimer of Mr 125,000) using affinity labeling methods and periodate-oxidized adenosine, ATP, and tRNA(Asp). It is shown that the number of periodate-oxidized derivatives which can be bound to the synthetase via Schiff's base formation with epsilon-NH2 groups of lysine residues exceeds the stoichiometry of specific substrate binding. Furthermore, it is found that the enzymatic activities are not completely abolished, even for high incorporation levels of the modified substrates. The tRNA(Asp) aminoacylation reaction is more sensitive to labeling than is the ATP-PPi exchange one; for enzyme preparations modified with oxidized adenosine or ATP this activity remains unaltered. These results demonstrate the absence of a specific lysine residue directly involved in the catalytic activities of yeast aspartyl-tRNA synthetase. Comparative labeling experiments with oxidized ATP were run with several other aminoacyl-tRNA synthetases. Residual ATP-PPi exchange and tRNA aminoacylation activities measured in each case on the modified synthetases reveal different behaviors of these enzymes when compared to that of aspartyl-tRNA synthetase. When tested under identical experimental conditions, pure isoleucyl-, methionyl-, threonyl- and valyl-tRNA synthetases from E. coli can be completely inactivated for their catalytic activities; for E. coli alanyl-tRNA synthetase only the tRNA charging activity is affected, whereas yeast valyl-tRNA synthetase is only partly inactivated. The structural significance of these experiments and the occurrence of essential lysine residues in aminoacyl-tRNA synthetases are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)
利用亲和标记法以及高碘酸盐氧化的腺苷、ATP和天冬氨酸tRNA，在面包酵母天冬氨酰-tRNA合成酶（一种Mr为125,000的α2二聚体）的催化位点寻找必需的赖氨酸残基。结果表明，通过与赖氨酸残基的ε-NH2基团形成席夫碱而能够与合成酶结合的高碘酸盐氧化衍生物的数量超过了特异性底物结合的化学计量。此外，还发现即使修饰底物的掺入水平很高，酶活性也不会完全丧失。天冬氨酸tRNA氨酰化反应比ATP-PPi交换反应对标记更敏感；对于用氧化腺苷或ATP修饰的酶制剂，该活性保持不变。这些结果表明，酵母天冬氨酰-tRNA合成酶的催化活性中不存在直接参与的特定赖氨酸残基。用氧化ATP进行了与其他几种氨酰-tRNA合成酶的比较标记实验。在每种情况下对修饰后的合成酶测定的残留ATP-PPi交换和tRNA氨酰化活性表明，与天冬氨酰-tRNA合成酶相比，这些酶有不同的行为。在相同实验条件下进行测试时，来自大肠杆菌的纯异亮氨酰-、甲硫氨酰-、苏氨酰-和缬氨酰-tRNA合成酶的催化活性可被完全灭活；对于大肠杆菌丙氨酰-tRNA合成酶，只有tRNA充电活性受到影响，而酵母缬氨酰-tRNA合成酶只是部分失活。讨论了这些实验的结构意义以及氨酰-tRNA合成酶中必需赖氨酸残基的存在情况。（摘要截短于250字）
