Aminoacyl-tRNA synthetases (aaRS) ensure the faithful transmission of genetic information in all living cells. The 24 known aaRS families are divided into 2 structurally distinct classes (class I and class II), each featuring a catalytic domain with a common fold that binds ATP, amino acid, and the 3'-terminus of tRNA. In a common two-step reaction, each aaRS first uses the energy stored in ATP to synthesize an activated aminoacyl adenylate intermediate. In the second step, either the 2'- or 3'-hydroxyl oxygen atom of the 3'-A76 tRNA nucleotide functions as a nucleophile in synthesis of aminoacyl-tRNA. Ten of the 24 aaRS families are unable to distinguish cognate from noncognate amino acids in the synthetic reactions alone. These enzymes possess additional editing activities for hydrolysis of misactivated amino acids and misacylated tRNAs, with clearance of the latter species accomplished in spatially separate post-transfer editing domains. A distinct class of trans-acting proteins that are homologous to class II editing domains also perform hydrolytic editing of some misacylated tRNAs. Here we review essential themes in catalysis with a view toward integrating the kinetic, stereochemical, and structural mechanisms of the enzymes. Although the aaRS have now been the subject of investigation for many decades, it will be seen that a significant number of questions regarding fundamental catalytic functioning still remain unresolved.

Hydrolytic editing activities are present in aminoacyl-tRNA synthetases possessing reduced amino acid discrimination in the synthetic reactions. Post-transfer hydrolysis of misacylated tRNA in class I editing enzymes occurs in a spatially separate domain inserted into the catalytic Rossmann fold, but the location and mechanisms of pre-transfer hydrolysis of misactivated amino acids have been uncertain. Here, we use novel kinetic approaches to distinguish among three models for pre-transfer editing by Escherichia coli isoleucyl-tRNA synthetase (IleRS). We demonstrate that tRNA-dependent hydrolysis of noncognate valyl-adenylate by IleRS is largely insensitive to mutations in the editing domain of the enzyme and that noncatalytic hydrolysis after release is too slow to account for the observed rate of clearing. Measurements of the microscopic rate constants for amino acid transfer to tRNA in IleRS and the related valyl-tRNA synthetase (ValRS) further suggest that pre-transfer editing in IleRS is an enzyme-catalyzed activity residing in the synthetic active site. In this model, the balance between pretransfer and post-transfer editing pathways is controlled by kinetic partitioning of the noncognate aminoacyl-adenylate. Rate constants for hydrolysis and transfer of a noncognate intermediate are roughly equal in IleRS, whereas in ValRS transfer to tRNA is 200-fold faster than hydrolysis. In consequence, editing by ValRS occurs nearly exclusively by post-transfer hydrolysis in the editing domain, whereas in IleRS both pre-and post-transfer editing are important. In both enzymes, the rates of amino acid transfer to tRNA are similar for cognate and noncognate aminoacyl-adenylates, providing a significant contrast with editing DNA polymerases.

Methanogenic archaea possess unusual seryl-tRNA synthetase (SerRS), evolutionarily distinct from the SerRSs found in other archaea, eucaryotes and bacteria. The two types of SerRSs show only minimal sequence similarity, primarily within class II conserved motifs 1, 2 and 3. Here, we report a 2.5 Å resolution crystal structure of the atypical methanogenic Methanosarcina barkeri SerRS and its complexes with ATP, serine and the nonhydrolysable seryl-adenylate analogue 5 0 -O-(N-serylsulfamoyl)adenosine. The structures reveal two idiosyncratic features of methanogenic SerRSs: a novel N-terminal tRNA-binding domain and an active site zinc ion. The tetra-coordinated Zn 2 þ ion is bound to three conserved protein ligands (Cys306, Glu355 and Cys461) and binds the amino group of the serine substrate. The absolute requirement of the metal ion for enzymatic activity was confirmed by mutational analysis of the direct zinc ion ligands. This zinc-dependent serine recognition mechanism differs fundamentally from the one employed by the bacterial-type SerRSs. Consequently, SerRS represents the only known aminoacyl-tRNA synthetase system that evolved two distinct mechanisms for the recognition of the same amino-acid substrate.

Background: Error-prone aminoacyl-tRNA synthetases clear noncognate aminoacyl-adenylates and misacylated tRNAs within synthetic and editing sites, respectively. Results: Product release limits the rate of post-transfer editing by leucyl-tRNA synthetase. Conclusion: Kinetic partitioning of misacylated tRNA determines the relative contribution of cis and trans editing. Significance: In contrast to DNA polymerases, error correction in class I tRNA synthetases relies on substrate selection by the editing site.

Glutaminyl-tRNA synthetase generates Gln-tRNA Gln 10 7 -fold more efficiently than Glu-tRNA Gln and requires tRNA to synthesize the activated aminoacyl adenylate in the first step of the reaction. To examine the role of tRNA in amino acid activation more closely, several assays employing a tRNA analog in which the 2-OH group at the 3-terminal A76 nucleotide is replaced with hydrogen (tRNA 2H Gln ) were developed. These experiments revealed a 10 4 -fold reduction in k cat /K m in the presence of the analog, suggesting a direct catalytic role for tRNA in the activation reaction. The catalytic importance of the A76 2-OH group in aminoacylation mirrors a similar role for this moiety that has recently been demonstrated during peptidyl transfer on the ribosome. Unexpectedly, tracking of Gln-AMP formation utilizing an ␣-32 P-labeled ATP substrate in the presence of tRNA 2H Gln showed that AMP accumulates 5-fold more rapidly than Gln-AMP. A cold-trapping experiment revealed that the nonenzymatic rate of Gln-AMP hydrolysis is too slow to account for the rapid AMP formation; hence, the hydrolysis of Gln-AMP to form glutamine and AMP must be directly catalyzed by the GlnRS⅐tRNA 2H Gln complex. This hydrolysis of glutaminyl adenylate represents a novel reaction that is directly analogous to the pre-transfer editing hydrolysis of noncognate aminoacyl adenylates by editing synthetases such as isoleucyl-tRNA synthetase. Because glutaminyltRNA synthetase does not possess a spatially separate editing domain, these data demonstrate that a pre-transfer editing-like reaction can occur within the synthetic site of a class I tRNA synthetase.The specificity of protein synthesis depends upon the fidelity of aminoacyl-tRNA synthetases (aaRS).1 These enzymes attach amino acids to the 3Ј terminus of transfer RNAs in a two-step reaction (1). First, the amino acid is activated by reaction with ATP, to yield an aminoacyl adenylate intermediate and pyrophosphate. In the second step, one of the two hydroxyl oxygens of the 3Ј-terminal A76 nucleotide of tRNA attacks the carbonyl carbon of the adenylate, producing aminoacyl-tRNA with release of AMP. Each synthetase must discriminate among both structurally similar amino acids and tRNAs, selecting only the cognate species from cellular pools with an overall accuracy of approximately one error per 10 4 -10 5 codons (2). While the subsequent interaction of aminoacyl-tRNA with elongation factors may also provide some selection (3), it is clear that the specificity of protein synthesis primarily arises from the tRNA synthetase-mediated step.It is well established that some tRNA synthetases are unable to accurately discriminate among chemically similar amino acids based solely on interactions made in the synthetic active site (reviewed in Ref. 4). These enzymes possess an additional hydrolytic activity for deacylation of misaminoacylated tRNAs. This reaction occurs in a second active site that effectively excludes correctly aminoacylated products. For example, in IleRS the synthetic active site canno...

Aminoacyl-tRNA synthetases, a group of enzymes catalyzing aminoacyl-tRNA formation, may possess inherent editing activity to clear mistakes arising through the selection of non-cognate amino acid. It is generally assumed that both editing substrates, non-cognate aminoacyl-adenylate and misacylated tRNA, are hydrolyzed at the same editing domain, distant from the active site. Here, we present the first example of an aminoacyl-tRNA synthetase (seryl-tRNA synthetase) that naturally lacks an editing domain, but possesses a hydrolytic activity toward non-cognate aminoacyl-adenylates. Our data reveal that tRNA-independent pre-transfer editing may proceed within the enzyme active site without shuttling the non-cognate aminoacyl-adenylate intermediate to the remote editing site.

Steady-state and transient kinetic analyses of glutaminyl-tRNA synthetase (GlnRS) reveal that the enzyme discriminates against noncognate glutamate at multiple steps during the overall aminoacylation reaction. A major portion of the selectivity arises in the amino acid activation portion of the reaction, whereas the discrimination in the overall two-step reaction arises from very weak binding of noncognate glutamate. Further transient kinetics experiments showed that tRNA Gln binds to GlnRS ϳ60-fold weaker when noncognate glutamate is present and that glutamate reduces the association rate of tRNA with the enzyme by 100-fold. These findings demonstrate that amino acid and tRNA binding are interdependent and reveal an important additional source of specificity in the aminoacylation reaction. Crystal structures of the GlnRS⅐tRNA complex bound to either amino acid have previously shown that glutamine and glutamate bind in distinct positions in the active site, providing a structural basis for the amino acid-dependent modulation of tRNA affinity. Together with other crystallographic data showing that ligand binding is essential to assembly of the GlnRS active site, these findings suggest a model for specificity generation in which required induced-fit rearrangements are significantly modulated by the identities of the bound substrates.The high fidelity of protein synthesis in living cells arises as a consequence of specificity at three distinct steps in the pathway: aminoacyl-tRNA formation, selection of aminoacyltRNA by elongation factor Tu, and ribosomal proofreading of the codon-anticodon interaction (1-3). Accuracy at the first aminoacyl-tRNA synthesis step is accomplished by the aminoacyl-tRNA synthetases, which attach amino acids to the corresponding tRNA species in a two-step reaction. Amino acids are first activated to their corresponding adenylates by harnessing the energy of ATP hydrolysis, generating pyrophosphate. In the second step, the oxygen nucleophile at the 2Ј-or 3Ј-ribose position of the 3Ј-terminal A76 tRNA nucleotide attacks the carbonyl carbon of the mixed anhydride intermediate, forming aminoacyl-tRNA with release of AMP (1). Formation of only cognate aminoacyl-tRNA species requires that the enzymes be specific for both amino acid and tRNA substrates.Although all tRNAs possess largely similar L-shaped tertiary structures, significant capacity for discrimination among these species arises from direct recognition of base-specific functional groups at key nucleotide positions (identity determinants), as well as by an indirect readout process in which selective enzyme interactions are made with groups in the sugar-phosphate backbone or nonspecific portions of the bases (4). The existence of 20 parallel aminoacyl-tRNA systems in vivo promotes further discrimination by competition (5). In contrast, amino acid specificity relies on a much smaller number of possible interactions between the enzyme and substrate; moreover, many of the amino acids possess similar structural and chemical properties. In s...

The fidelity of protein synthesis depends on the capacity of aminoacyl-tRNA synthetases (AARSs) to couple only cognate amino acidtRNA pairs. If amino acid selectivity is compromised, fidelity can be ensured by an inherent AARS editing activity that hydrolyses mischarged tRNAs. Here, we show that the editing activity of Escherichia coli leucyl-tRNA synthetase (EcLeuRS) is not required to prevent incorrect isoleucine incorporation. Rather, as shown by kinetic, structural and in vivo approaches, the prime biological function of LeuRS editing is to prevent mis-incorporation of the non-standard amino acid norvaline. This conclusion follows from a reassessment of the discriminatory power of LeuRS against isoleucine and the demonstration that a LeuRS editing-deficient E. coli strain grows normally in high concentrations of isoleucine but not under oxygen deprivation conditions when norvaline accumulates to substantial levels. Thus, AARS-based translational quality control is a key feature for bacterial adaptive response to oxygen deprivation. The non-essential role for editing under normal bacterial growth has important implications for the development of resistance to antimicrobial agents targeting the LeuRS editing site.