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本文用3′-末端氧化的tRNA~(Leu)(tRNA_(ox)~(Leu))专一地标记了大肠杆菌亮氨酰-tRNA合成酶(LeuRS)。合成酶标记上等摩尔tRNA_(ox)~(Leu)后,氨酰化活力完全丧失,同时氨基酸活化活力也失去80％。这样失活反应是通过tRNA_(ox)~(Leu)的3′-末端羰基与亮氨酰-tRNA合成酶活性中心或其附近的赖氨酸残基的ε-氨基形成Schiff碱实现的。Schiff碱经硼氢氰化钠还原后稳定。酶与tRNA_(ox)~(Leu)的共价复合物经牛胰核糖核酸酶充分水解后,其活化氨基酸的活力及动力学常数基本恢复到原来水平,而氨基酰化活力未恢复。表明了该合成酶的两种活性中心相互关联又有区别。
In this paper, E. coli leucyl-tRNA synthetase (LeuRS) was specifically labeled with 3’-terminal oxidized tRNA ~ (Leu) (tRNA_ (ox) ~ (Leu)). After the same amount of tRNA (ox) ~ (Leu) was labeled by the synthetase, the aminoacylation activity was completely lost and the amino acid activation activity was also lost by 80%. Such an inactivation reaction is achieved by forming a Schiff base from the 3’-terminal carbonyl of tRNA_ (ox) ~ (Leu) to the ε-amino group of the lysine residue at or near the active site of leucyl-tRNA synthetase. Schiff base is stabilized by sodium borohydride reduction. After the covalent complex of enzyme and tRNA_ (ox) ~ (Leu) was fully hydrolyzed by bovine pancreatic ribonuclease, the viability and kinetic constants of activated amino acids returned to their original level, but the aminoacylation activity did not recover. This shows that the two active centers of the synthetase are related to each other.