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.

Pupylation is a post-translational protein modification occurring in mycobacteria and other actinobacteria that is functionally analogous to ubiquitination. Here, we report the crystal structures of the modification enzymes involved in this pathway, the Pup ligase PafA and the depupylase/deamidase Dop. Both feature a larger N-terminal domain consisting of a central β-sheet packed against a cluster of helices, a fold characteristic for carboxylate-amine ligases, and a smaller C-terminal domain unique to PafA/Dop members. The active site is located on the concave surface of the β-sheet with the nucleotide bound in a deep pocket. A conserved groove leading into the active site could play a role in Pup-binding. NMR and biochemical experiments determine the region of Pup that interacts with PafA and Dop. Structural data and mutational studies identify crucial residues for catalysis of both enzymes.

In Mycobacterium tuberculosis, the enzyme PafA is responsible for the activation and conjugation of the proteasometargeting molecule Pup to protein substrates. As the proteasomal pathway has been shown to be vital to the persistence of M. tuberculosis, understanding the reaction mechanism of PafA is critical to the design of antituberculous agents. In this study, we have developed novel techniques to study the activity of PafA and have characterized fundamental features of the reaction mechanism. We show that PafA catalyzes a two-step reaction mechanism proceeding through a ␥-glutamyl phosphate-mixed anhydride intermediate that is formed on the Cterminal glutamate of Pup before transfer of Pup to the substrate acceptor lysine. SDS-PAGE analysis of formation of the phosphorylated intermediate revealed that the rate of Pup activation matched the maximal steady-state rate of product formation in the overall reaction and suggested that Pup activation was rate-limiting when all substrates were present at saturating concentrations. Following activation, both ADP and the phosphorylated intermediate remained associated with the enzyme awaiting nucleophilic attack by a lysine residue of the target protein. The PafA reaction mechanism appeared to be noticeably biased toward the stable activation of Pup in the absence of additional substrate and required very low concentrations of ATP and Pup relative to other carboxylate-amine/ ammonia ligase family members. The bona fide nucleophilic substrate PanB showed a 3 orders of magnitude stronger affinity than free lysine, promoting Pup conjugation to occur close to the rate limit of activation with physiologically relevant concentrations of substrate.

The selection of tRNAs by their cognate aminoacyl-tRNA synthetases is critical for ensuring the fidelity of protein synthesis. While nucleotides that comprise tRNA identity sets have been readily identified, their specific role in the elementary steps of aminoacylation is poorly understood. By use of a rapid kinetics analysis employing mutants in tRNA(His) and its cognate aminoacyl-tRNA synthetase, the role of tRNA identity in aminoacylation was investigated. While mutations in the tRNA anticodon preferentially affected the thermodynamics of initial complex formation, mutations in the acceptor stem or the conserved motif 2 loop of the tRNA synthetase imposed a specific kinetic block on aminoacyl transfer and decreased tRNA-mediated kinetic control of amino acid activation. The mechanistic basis of tRNA identity is analogous to fidelity control by DNA polymerases and the ribosome, whose reactions also demand high accuracy.

All histidine tRNA molecules have an extra nucleotide, G-1, at the 59 end of the acceptor stem. In bacteria, archaea, and eukaryotic organelles, G-1 base pairs with C73, while in eukaryotic cytoplasmic tRNA His , G-1 is opposite A73. Previous studies of Escherichia coli histidyl-tRNA synthetase (HisRS) have demonstrated the importance of the G-1:C73 base pair to tRNA His identity. Specifically, the 59-monophosphate of G-1 and the major groove amine of C73 are recognized by E. coli HisRS; these individual atomic groups each contribute ; ;4 kcal/mol to transition state stabilization. In this study, two chemically synthesized 24-nucleotide RNA microhelices, each of which recapitulates the acceptor stem of either E. coli or Saccharomyces cervisiae tRNA His , were used to facilitate an atomic group ''mutagenesis'' study of the ÿ1:73 base pair recognition by S. cerevisiae HisRS. Compared with E. coli HisRS, microhelixHis is a much poorer substrate relative to full-length tRNA His for the yeast enzyme. However, the data presented here suggest that, similar to the E. coli system, the 59 monophosphate of yeast tRNA His is critical for aminoacylation by yeast HisRS and contributes ; ;3 kcal/mol to transition state stability. The primary role of the unique ÿ1:73 base pair of yeast tRNA His appears to be to properly position the critical 59 monophosphate for interaction with the yeast enzyme. Our data also suggest that the eukaryotic HisRS/tRNA His interaction has coevolved to rely less on specific major groove interactions with base atomic groups than the bacterial system.