TRP TRNA LIGASE--X-RAY STUDIES OF THE CATALYTIC CYCLE

我们的目标是关联在氨酰化过程中的构象变化 具有特异性识别和底物转化的tRNA。我们 希望测试构象变化的具体预测 B-色氨酰-tRNA合成酶(TrpRS)的观察 从无配体酶到Trp-5‘AMP复合体位置的探讨 适合于酰基转移的tRNA反密码子结合部位，相对于 另一种单体的活性部位。为此，我们将解决新的 TrpRS与同源tRNA和与ATP的复合体的X射线结构。 在前一个资金周期中，我们解决了无配体的酶和 色氨酸络合物；一种活性基态三元络合物 使用ATP和特定物种的抑制剂吲哚霉素；天然的 腺化中间体Trp-5‘AMP和产物色氨酰-2’3‘-ATP。 我们将延长色氨酸-5‘AMP的分辨率和实验阶段 比1.7埃单位好的衍射极限， 为了精确地确定N-之间的侧链堆积相互作用 Rossmann-折叠结构域的末端螺旋两个单体结构域， 它显然将活性部位的行为与远端反密码子偶联 通过Ile 16结合位点。Ile 16将突变为缬氨酸、亮氨酸和 丙氨酸来检验这一假设，即这种残基与小分子结合 包含反密码子结合位点的结构域。我们会完成的 从上面的列表中提炼出每个相关的结构，按顺序 以产生氨基酸活化的结构反应轮廓，并 TRNA的酰化。特别值得注意的是二聚体的变化 通过将tRNA的受体茎与一个结合而引入的界面 活性位点和反密码子连接到另一个单体上的一个位点。至 解释反密码子突变体是如何抑制色氨酸的 ，我们将把该复合体与以前获得的 为了GlnRS。 原核生物TrpRS是潜在的有价值的抗感染靶点 药物发现，由于可获得原核生物特异的 抑制剂，吲哚霉素。我们将研究高增长的结构性基础。 通过比较几种此类络合物和碱基的亲和力结合 通过将我们的结构分析扩展到古细菌 和真核细胞的TrpRSs。

Our goal is to correlate conformation changes during aminoacylation of tRNA by with specific recognition and substrate transformations. We wish to test the specific prediction that conformational changes observed for B stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) on proceeding from ligand-free enzyme to the Trp-5' AMP complex position the tRNA anticodon-binding site suitably for acyl-transfer, relative to the active site of the other monomer. To this end, we will solve new X-ray structures of TrpRS complexes with the cognate tRNA and with ATP. During the previous funding cycle we solved the ligand-free enzyme and complexes with tryptophan; an activated ground-state ternary complex with ATP and the species-specific inhibitor, indolmycin; the natural adenylate intermediate, Trp-5'AMP, and a product, tryptophanyl-2'3'-ATP. We will extend the resolution and experimental phases for the Trp-5' AMP complex to its diffraction limit which better than 1.7 Angstrom units, to precisely specific sidechain packing interactions between the N- terminal helix of the Rossmann-fold domain two domains of the monomer, which apparently couple active-site behavior to the distal anticodon binding site via Ile 16. Ile 16 will be mutated to valine, leucine, and alanine to test that hypothesis that this residue couples the small domain containing the anticodon-binding site to. We will finish refining each of the relevant structures from the above list, in order to produce a structural reaction profile for aminoacid activation and acylation of tRNA. Of special interest will be changes at the dimer interface introduced by binding the acceptor stem of the tRNA to one active site and the anticodon to a site on the other monomer. To explain how suppression with tryptophan occurs with an anticodon mutant of trRNA gin, we will compare the complex with the previously obtained for GlnRS. Prokaryote TrpRS is a potentially valuable target for anti-infective drug discovery, owing to the availability of a prokaryote-specific inhibitor, indolmycin. We will examine the structural bases for high- affinity binding by comparison of several such complexes, and the bases for specificity by extending our structural analysis to archebacterial and eukaryotic TrpRSs.