From the Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7260.
J Biol Chem. 2013 Nov 29;288(48):34736-45. doi: 10.1074/jbc.M113.510958. Epub 2013 Oct 20.
Tryptophanyl-tRNA Synthetase (TrpRS) Urzyme (fragments A and C), a 130-residue construct containing only secondary structures positioning the HIGH and KMSKS active site signatures and the specificity helix, accelerates tRNA(Trp) aminoacylation with ∼10-fold specificity toward tryptophan, relative to structurally related tyrosine. We proposed that including the 76-residue connecting peptide 1 insertion (Fragment B) might enhance tryptophan affinity and hence amino acid specificity, because that subdomain constrains the orientation of the specificity helix. We test that hypothesis by characterizing two new constructs: the catalytic domain (fragments A-C) and the Urzyme supplemented with the anticodon-binding domain (fragments A, C, and D). The three constructs, together with the full-length enzyme (fragments A-D), comprise a factorial experiment from which we deduce individual and combined contributions of the two modules to the steady-state kinetics parameters for tryptophan-dependent (32)PPi exchange, specificity for tryptophan versus tyrosine, and aminoacylation of tRNA(Trp). Factorial design directly measures the energetic coupling between the two more recent modules in the contemporary enzyme and demonstrates its functionality. Combining the TrpRS Urzyme individually in cis with each module affords an analysis of long term evolution of amino acid specificity and tRNA aminoacylation, both essential for expanding the genetic code. Either module significantly enhances tryptophan activation but unexpectedly eliminates amino acid specificity for tryptophan, relative to tyrosine, and significantly reduces tRNA aminoacylation. Exclusive dependence of both enhanced functionalities of full-length TrpRS on interdomain coupling energies between the two new modules argues that independent recruitment of connecting peptide 1 and the anticodon-binding domain during evolutionary development of Urzymes would have entailed significant losses of fitness.
色氨酰-tRNA 合成酶（TrpRS）酶（片段 A 和 C）是一种 130 残基的构建体，仅包含二级结构，定位 HIGH 和 KMSKS 活性位点特征和特异性螺旋，相对于结构相关的酪氨酸，可将 tRNA（Trp）氨酰化的特异性提高约 10 倍。我们提出，包含 76 残基连接肽 1 插入（片段 B）可能会增强色氨酸亲和力，从而提高氨基酸特异性，因为该亚结构域限制了特异性螺旋的取向。我们通过表征两个新的构建体来验证该假设：催化结构域（片段 A-C）和补充反密码子结合结构域的酶（片段 A、C 和 D）。这三个构建体与全长酶（片段 A-D）一起构成了一个因子实验，我们从中推断出两个模块对依赖色氨酸的（32）PPi 交换的稳态动力学参数、色氨酸与酪氨酸的特异性以及 tRNA（Trp）的氨酰化的个体和组合贡献。因子设计直接测量了当代酶中两个最近模块之间的能量偶联，并证明了其功能。将 TrpRS 酶单独顺式组合，可以分析氨基酸特异性和 tRNA 氨酰化的长期进化，这对于扩展遗传密码至关重要。两个模块中的任何一个都能显著增强色氨酸的激活，但出乎意料的是，与酪氨酸相比，对色氨酸的氨基酸特异性降低，并且显著降低 tRNA 的氨酰化。全长 TrpRS 的这两种增强功能都完全依赖于两个新模块之间的域间耦合能量，这表明在 Urzymes 的进化发展过程中，连接肽 1 和反密码子结合结构域的独立募集将导致适应性显著丧失。
