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

点击翻译按钮获取中文摘要

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.