Wu M X, Filley S J, Xiong J, Lee J J, Hill K A
Department of Biochemistry, Loma Linda University School of Medicine, California 92350.
Biochemistry. 1994 Oct 11;33(40):12260-6. doi: 10.1021/bi00206a032.
Alanyl-tRNA synthetase (AlaRS) from Escherichia coli is a multimeric enzyme that catalyzes the esterification of alanine to tRNA(Ala) in the ATP-dependent aminoacylation reaction. The functional binding of all three substrates follows Michaelis-Menten kinetics. The role of cysteines in this enzyme has been evaluated via modification of these residues with p-(hydroxymercuri)phenylsulfonic acid, monobromobimane, and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). The former two reagents induce nearly complete inactivation of AlaRS aminoacylation activity and the release of all tightly bound zinc. In the case of mild DTNB treatment, only two of the six cysteines in AlaRS are modified, with release of all zinc and partial loss of aminoacylation activity. These experiments indicate the importance of one or more cysteines, other than those thought to be coordinated with zinc, in the aminoacylation reaction. Substitution of each of the cysteine residues outside the zinc-binding motif with serine does not disrupt zinc binding. However, the cysteine most removed in primary sequence from the active site (Cys665) is identified as important in the aminoacylation step. Mutation of Cys665 to serine induces a 120-fold decrease in the catalytic efficiency of this enzyme, primarily through a kcat effect, and introduces sigmoidal kinetics (nH = 1.8) with respect to the RNA substrate. The results demonstrate that a simple manipulation in the C-terminal region can introduce positive cooperativity in this otherwise noncooperative enzyme.
来自大肠杆菌的丙氨酰 - tRNA合成酶（AlaRS）是一种多聚体酶，它在依赖ATP的氨酰化反应中催化丙氨酸与tRNA（Ala）的酯化反应。所有三种底物的功能性结合遵循米氏动力学。通过用对（羟基汞）苯磺酸、单溴联苯胺和5,5'-二硫代双（2-硝基苯甲酸）（DTNB）修饰这些残基，评估了该酶中半胱氨酸的作用。前两种试剂几乎完全使AlaRS的氨酰化活性失活，并释放出所有紧密结合的锌。在轻度DTNB处理的情况下，AlaRS的六个半胱氨酸中只有两个被修饰，锌全部释放，氨酰化活性部分丧失。这些实验表明，在氨酰化反应中，除了那些被认为与锌配位的半胱氨酸外，一个或多个半胱氨酸也很重要。用丝氨酸取代锌结合基序外的每个半胱氨酸残基不会破坏锌的结合。然而，在一级序列中与活性位点距离最远的半胱氨酸（Cys665）被确定在氨酰化步骤中很重要。将Cys665突变为丝氨酸会导致该酶的催化效率下降120倍，主要是通过kcat效应，并引入了关于RNA底物的S形动力学（nH = 1.8）。结果表明，在C端区域进行简单操作可以在这种原本无协同性的酶中引入正协同性。
