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

TRP TRNA 连接酶--催化循环的 X 射线研究

• 批准号: 6018930
• 负责人: Charles W. Carter
• 金额: $ 22.19万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-15 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018930
• 关键词: Bacillus stearothermophilus; SDS polyacrylamide gel electrophoresis; X ray crystallography; active sites; acylation; adenosine triphosphate; alanine; aminoacid tRNA ligase; aminoacyl tRNA; computer simulation; conformation; drug discovery /isolation; enzyme mechanism; enzyme structure; leucine; molecular dynamics; protein structure function; tryptophan; valine

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.

我们的目标是将构象变化相关联。 通过特定识别和底物转换。 我们 希望测试构象变化的具体预测 观察到b stearothothormophilus色氨酸-TRNA合成酶（TRPRS） 从无配体酶到TRP-5'AMP复合位置进行 tRNA反物质结合位点适当用于酰基转移，相对于 另一个单体的活跃部位。 为此，我们将解决新的 TRPRS复合物与同源tRNA和ATP的X射线结构。 在上一个资金周期中，我们解决了无配体酶和 带有色氨酸的复合物；活化的地下三元络合物 带有ATP和物种特异性抑制剂吲哚霉素；自然 腺苷酸中间体，TRP-5'AMP和Therpophanyl-2'3'-ATP产物。 我们将扩展TRP-5'AMP的分辨率和实验阶段 复杂到其衍射极限，大于1.7埃斯特罗姆单元， 确切特定特定的Sidechain填料相互作用 Rossmann-fold域的终端螺旋，单体的两个域， 显然将主动位点的行为与远端反登起 通过ILE 16的结合位点。ILE 16将突变为Valine，Leucine和 丙氨酸以检验该残基伴侣小的假设 包含反对多子结合位点的域。 我们将完成 从上面的列表中完善每个相关结构，按顺序 产生结构性反应特征，以进行氨基酸激活和 tRNA的酰化。 特别感兴趣的是二聚体的变化 通过将tRNA的受体茎与一个结合到一个 活性位点和另一个单体上的位点的反密码子。 到 说明色氨酸抑制是如何用反量产突变体发生的 trrna杜松子酒，我们将比较复合物与先前获得的 对于glnrs。 原核生物TRPR是反感染的潜在有价值的目标 药物发现，由于原核生物特异性的可用性 抑制剂，吲哚霉素。 我们将检查高的结构基础 通过比较几个这样的复合物和碱基的亲和力结合 通过将我们的结构分析扩展到弓形细菌来进行特异性 和真核生物trprss。