Jakubowski H
Biochim Biophys Acta. 1978 Dec 21;521(2):584-96. doi: 10.1016/0005-2787(78)90300-3.
As a continuation of our studies on plant (yellow lupin, Lupinus luteus) aminoacyl-tRNA synthetases we describe here formation and some properties of valyl-tRNA synthetase-bound valyl adenylate (EVal(Val-AMP)) and seryl-tRNA synthetase-bound seryl adenylate (ESer(Ser-AMP)). Valyl-tRNA synthetase-bound valyl adenylate was detected and isolated by several approaches in the pH range 6--10. In that range inorganic pyrophosphatase increases the amount of valyl adenylate by factor 1.8 regardless of pH. 50% of valine from the EVal(Val-AMP) complex isolated by Sephadex G-100 gel filtration was transferred to tRNA with a rate constant greater than 4 min-1 (pH 6.2, 10 degrees C). The ratio of valine to AMP in the enzyme-bound valyl adenylate is 1 : 1 and it is not changed by the presence of periodate-oxidized tRNA. In contrast to enzyme-bound valyl adenylate, formation of ESer(Ser-AMP) is very sensitive to pH. Inorganic pyrophosphatase increases the amount of seryl adenylate by a factor 6 at pH 8.0 and 30 at pH 6.9 60% of serine from the ESer(Ser-AMP) complex was transferred to tRNA with a rate constant greater than 4 min-1 (pH 8.0, 0 degrees C). The ratio of serine to AMP in the enzyme-bound seryl adenylate is 1 : 1. The rate of synthesis of the enzyme-bound aminoacyl adenylates was measured by ATP-PPi exchange. Michaelis constants for the substrates of valyl-tRNA and seryl-tRNA synthetases in ATP-PPi exchange were determined. Effects of pH, MgCl2 and KCl on the initial velocity of aminoacyl adenylate formation are described. For comparison, catalytic indices in the aminoacylation reactions catalyzed by both lupin enzymes are given and effects of pH, MgCl2 and KCl on tRNA aminoacylation are presented as well. Under some conditions, e.g. at low pH or high salt concentration, lupin valyl-tRNA and seryl-tRNA synthetase are active exclusively in ATP-PPi exchange reaction.
作为我们对植物（黄羽扇豆，Lupinus luteus）氨酰 - tRNA合成酶研究的延续，我们在此描述缬氨酰 - tRNA合成酶结合的缬氨酰腺苷酸（EVal(Val - AMP)）和丝氨酰 - tRNA合成酶结合的丝氨酰腺苷酸（ESer(Ser - AMP)）的形成及一些特性。通过多种方法在pH值6 - 10范围内检测并分离出缬氨酰 - tRNA合成酶结合的缬氨酰腺苷酸。在此范围内，无机焦磷酸酶使缬氨酰腺苷酸的量增加1.8倍，与pH值无关。通过Sephadex G - 100凝胶过滤分离得到的EVal(Val - AMP)复合物中，50%的缬氨酸以大于4 min⁻¹的速率常数（pH 6.2，10℃）转移至tRNA。酶结合的缬氨酰腺苷酸中缬氨酸与AMP的比例为1 : 1，且高碘酸盐氧化的tRNA的存在不会改变该比例。与酶结合的缬氨酰腺苷酸不同，ESer(Ser - AMP)的形成对pH值非常敏感。在pH 8.0时，无机焦磷酸酶使丝氨酰腺苷酸的量增加6倍，在pH 6.9时增加30倍。ESer(Ser - AMP)复合物中60%的丝氨酸以大于4 min⁻¹的速率常数（pH 8.0，0℃）转移至tRNA。酶结合的丝氨酰腺苷酸中丝氨酸与AMP的比例为1 : 1。通过ATP - PPi交换测定酶结合的氨酰腺苷酸的合成速率。测定了缬氨酰 - tRNA和丝氨酰 - tRNA合成酶在ATP - PPi交换中底物的米氏常数。描述了pH值、MgCl₂和KCl对氨酰腺苷酸形成初始速度的影响。为作比较，给出了两种羽扇豆酶催化的氨酰化反应中的催化指数，并呈现了pH值、MgCl₂和KCl对tRNA氨酰化的影响。在某些条件下，例如在低pH值或高盐浓度下，羽扇豆缬氨酰 - tRNA和丝氨酰 - tRNA合成酶仅在ATP - PPi交换反应中具有活性。
