The crystal structure of Escherichia coli glutaminyl-tRNA synthetase (GlnRS) complexed with its cognate glutaminyl transfer RNA (tRNA(Gln] and adenosine triphosphate (ATP) has been derived from a 2.8 angstrom resolution electron density map and the known protein and tRNA sequences. The 63.4-kilodalton monomeric enzyme consists of four domains arranged to give an elongated molecule with an axial ratio greater than 3 to 1. Its interactions with the tRNA extend from the anticodon to the acceptor stem along the entire inside of the L of the tRNA. The complexed tRNA retains the overall conformation of the yeast phenylalanine tRNA (tRNA(Phe] with two major differences: the 3' acceptor strand of tRNA(Gln) makes a hairpin turn toward the inside of the L, with the disruption of the final base pair of the acceptor stem, and the anticodon loop adopts a conformation not seen in any of the previously determined tRNA structures. Specific recognition elements identified so far include (i) enzyme contacts with the 2-amino groups of guanine via the tRNA minor groove in the acceptor stem at G2 and G3; (ii) interactions between the enzyme and the anticodon nucleotides; and (iii) the ability of the nucleotides G73 and U1.A72 of the cognate tRNA to assume a conformation stabilized by the protein at a lower free energy cost than noncognate sequences. The central domain of this synthetase binds ATP, glutamine, and the acceptor end of the tRNA as well as making specific interactions with the acceptor stem.2+t is

Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system.

The refined crystal structure of Escherichia coli glutaminyl transfer RNA synthetase complexed with transfer RNA(Gln) and ATP reveals that the structure of the anticodon loop of the enzyme-bound tRNA(Gln) differs extensively from that of the known crystal structures of uncomplexed tRNA molecules. The anticodon stem is extended by two non-Watson-Crick base pairs, leaving the three anti-codon bases unpaired and splayed out to bind snugly into three separate complementary pockets in the protein. These interactions suggest that the entire anticodon loop provides essential sites for glutaminyl tRNA synthetase discrimination among tRNA molecules.

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The structure of Escherichia coli glutaminyl-tRNA synthetase complexed with tRNA2Gln and ATP refined at 2.5-A resolution reveals structural details of the catalytic center and allows description of the specific roles of individual amino acid residues in substrate binding and catalysis. The reactive moieties of the ATP and tRNA substrates are positioned within hydrogen-bonding distance of each other. Model-building has been used to position the glutamine substrate in an adjacent cavity with its reactive carboxylate adjacent to the alpha-phosphate of ATP; the interactions of the carboxyamide side chain suggest a structural rationale for the way in which the enzyme discriminates against glutamate. The binding site for a manganese ion has also been identified bridging the beta- and gamma-phosphates of the ATP. The well-known HIGH and KMSKS sequence motifs interact directly with each other as well as with the ATP, providing a structural rationale for their simultaneous conservation in all class I synthetases. The KMSKS loop adopts a well-ordered and catalytically productive conformation as a consequence of interactions made with the proximal beta-barrel domain. While there are no protein side chains near the reaction site that might function in acid-base catalysis, the side chains of two residues, His43 and Lys270, are positioned to assist in stabilizing the expected pentacovalent intermediate at the alpha-phosphate. Transfer of glutamine to the 3'-terminal tRNA ribose may well proceed by intramolecular catalysis involving proton abstraction by a phosphate oxygen atom of glutaminyl adenylate. Catalytic competence of the crystalline enzyme is directly shown by its ability to hydrolyze ATP and release pyrophosphate when crystals of the ternary complex are soaked in mother liquor containing glutamine.

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.

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