A substrate-assisted concerted mechanism for aminoacylation by a class II aminoacyl-tRNA synthetase.
Aminoacyl-tRNA synthetases (aaRS) join amino acids to their cognate transfer RNAs, establishing an essential coding relationship in translation. To investigate the mechanism of aminoacyl transfer in class II Escherichia coli histidyl-tRNA synthetase (HisRS), we devised a rapid quench assay. Under single turnover conditions with limiting tRNA, aminoacyl transfer proceeds at 18.8 s(-)(1), whereas in the steady state, the overall rate of aminoacylation is limited by amino acid activation to a rate of 3 s(-)(1). In vivo, this mechanism may serve to allow the size of amino acid pools and energy charge to control the rate of aminoacylation and thus protein synthesis. Aminoacyl transfer experiments using HisRS active site mutants and phosphorothioate-substituted adenylate showed that substitution of the nonbridging Sp oxygen of the adenylate decreased the transfer rate at least 10 000-fold, providing direct experimental evidence for the role of this group as a general base for the reaction. Other kinetic experiments revealed that the rate of aminoacyl transfer is independent of the interaction between the carboxyamide group of Gln127 and the alpha-carboxylate carbon, arguing against the formation of a tetrahedral intermediate during the aminoacyl transfer. These experiments support a substrate-assisted concerted mechanism for HisRS, a feature that may generalize to other aaRS, as well as the peptidyl transferase center.
Guth
Ethan
E
Department of Biochemistry, University of Vermont, Health Sciences Complex, Burlington, Vermont 05405, USA.
Connolly
Susan H
SH
Bovee
Michael
M
Francklyn
Christopher S
CS
D019943:Amino Acid Substitution / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D019281:Dimerization; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006643:Histidine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012354:RNA, Transfer, His / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013873:Thionucleotides / Q000737:chemistry / Q000378:metabolism; D046249:Transfer RNA Aminoacylation / Q000235:genetics
D029968:Escherichia coli Proteins; D012354:RNA, Transfer, His; D013873:Thionucleotides; C017817:adenosine 5'-(1-thio)triphosphate; D006643:Histidine-tRNA Ligase