title: RNA-assisted catalysis in a protein enzyme: The 2'-hydroxyl of tRNA(Thr) A76 promotes aminoacylation by threonyl-tRNA synthetase.
abstract: Aminoacyl-tRNA synthetases (aaRSs) join amino acids to 1 of 2 terminal hydroxyl groups of their cognate tRNAs, thereby contributing to the overall fidelity of protein synthesis. In class II histidyl-tRNA synthetase (HisRS) the nonbridging S(p)-oxygen of the adenylate is a potential general base for aminoacyl transfer. To test for conservation of this mechanism in other aaRSs and the role of terminal hydroxyls of tRNA in aminoacyl transfer, we investigated the class II Escherichia coli threonyl-tRNA synthetase (ThrRS). As with other class II aaRSs, the rate-determining step for ThrRS is amino acid activation. In ThrRS, however, the 2'-OH of A76 of tRNA(Thr) and a conserved active-site histidine (His-309) collaborate to catalyze aminoacyl transfer by a mechanism distinct from HisRS. Conserved residues in the ThrRS active site were replaced with alanine, and then the resulting mutant proteins were analyzed by steady-state and rapid kinetics. Nearly all mutants preferentially affected the amino acid activation step, with only a modest effect on aminoacyl transfer. By contrast, H309A ThrRS decreased transfer 242-fold and imposed a kinetic block to CCA accommodation. His-309 hydrogen bonds to the 2'-OH of A76, and substitution of the latter by hydrogen or fluorine decreased aminoacyl transfer by 763- and 94-fold, respectively. The proton relay mechanism suggested by these data to promote aminoacylation is reminiscent of the NAD(+)-dependent mechanisms of alcohol dehydrogenases and sirtuins and the RNA-mediated catalysis of the ribosomal peptidyl transferase center.
authors.affiliation: Cell and Molecular Biology Program, College of Medicine, Health Sciences Complex, 89 Beaumont Avenue, University of Vermont, Burlington, VT 05405, USA.
authors.forename: Anand
authors.initials: A
authors.lastname: Minajigi
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Francklyn
mesh_terms: D049148:Aminoacylation; D002384:Catalysis; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000378:metabolism; D012363:RNA, Transfer, Thr / Q000378:metabolism*; D013914:Threonine-tRNA Ligase / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D012363:RNA, Transfer, Thr; D017665:Hydroxyl Radical; D006639:Histidine; D013914:Threonine-tRNA Ligase