Park S J, Miller W T, Schimmel P
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1990 Oct 2;29(39):9212-8. doi: 10.1021/bi00491a015.
A 40 amino acid sequence of the unsolved structure of Escherichia coli alanine-tRNA synthetase is essential for tRNA binding and encodes an immunological determinant that cross-reacts with antibodies raised against a eukaryote (insect Bombyx mori) alanine enzyme. The secondary structure of this sequence is predicted to be an amphiphilic alpha-helix that includes one aspartyl and eight glutamyl side chain carboxyl groups. The antibody reactivity and the conformation of a synthetic peptide model of this region (Glu346 to Ser385) were investigated. In addition, double Arg----Gln and Leu----Ala substitutions were separately placed in the enzyme on the hydrophilic and hydrophobic face, respectively, of the predicted helix. These mutations conserve the polar/nonpolar character of each face and retain the potential for helix formation. Circular dichroism spectra of the synthetic peptide model demonstrate the potential for amphiphilic helix formation for the segment from Glu346 to Ser385. The behavior of the mutations in the enzyme, together with earlier data and immunological assays presented here, suggests that one face of the putative helix is an antigenic region of the surface of the enzyme where it contributes to the interaction with alanine tRNA and that the specific sequence of the helix is an important determinant of enzyme stability.
大肠杆菌丙氨酸 - tRNA合成酶未解析结构的一段40个氨基酸的序列对于tRNA结合至关重要，并且编码一个与针对真核生物（家蚕）丙氨酸酶产生的抗体发生交叉反应的免疫决定簇。该序列的二级结构预计为两亲性α - 螺旋，其中包括一个天冬氨酰侧链羧基和八个谷氨酰侧链羧基。研究了该区域（Glu346至Ser385）的合成肽模型的抗体反应性和构象。此外，分别在预测螺旋的亲水面和疏水面上对酶进行了双Arg→Gln和Leu→Ala替换。这些突变保留了每个面的极性/非极性特征，并保留了形成螺旋的潜力。合成肽模型的圆二色光谱证明了Glu346至Ser385片段形成两亲性螺旋的潜力。酶中突变的行为，连同本文给出的早期数据和免疫测定结果，表明推定螺旋的一个面是酶表面的一个抗原区域，在那里它有助于与丙氨酸tRNA相互作用，并且螺旋的特定序列是酶稳定性的一个重要决定因素。
