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In its tRNA acceptor end binding domain, the glutamyl-tRNA synthetase (GluRS) of Escherichia coli contains one atom of zinc that holds the extremities of a segment (Cys98-x-Cys100-x24-Cys125-x-His127) homologous to the Escherichia coli glutaminyl-tRNA synthetase (GlnRS) loop where a leucine residue stabilizes the peeled-back conformation of tRNAGln acceptor end. We report here that the GluRS zinc-binding region belongs to the novel SWIM domain family characterized by the signature C-x-C-xn-C-x-H (n = 6–25), and predicted to interact with DNA or proteins. In the presence of tRNAGlu, the GluRS C100Y variant has a lower affinity for l-glutamate than the wild-type enzyme, with Km and Kd values increased 12- and 20-fold, respectively. On the other hand, in the absence of tRNAGlu, glutamate binds with the same affinity to the C100Y variant and to wild-type GluRS. In the context of the close structural and mechanistic similarities between GluRS and GlnRS, these results indicate that the GluRS SWIM domain modulates glutamate binding to the active site via its interaction with the tRNAGlu acceptor arm. Phylogenetic analyses indicate that ancestral GluRSs had a strong zinc-binding site in their SWIM domain. Considering that all GluRSs require a cognate tRNA to activate glutamate, and that some of them have different or no putative zinc-binding residues in the corresponding positions, the properties of the C100Y variant suggest that the GluRS SWIM domains evolved to position correctly the tRNA acceptor end in the active site, thereby contributing to the formation of the glutamate binding site.

Discrimination factors (D) which are characteristic for discrimination between lysine and 19 naturally occuring non-cognate amino acids have been determined from kcat and Km values for native and phosphorylated lysyl-tRNA synthetases from yeast. Generally, both species of this class II aminoacyl-tRNA synthetase are considerably less specific than the class I synthetases specific for isoleucine, valine, tyrosine, and arginine. D values of the native enzyme are in the range 90–1700, D values of the phosphorylated species in the range 40–770. The phosphorylated enzyme acts faster and less accurately. In aminoacylation of tRNALys-C-C-A(2′NH2) discrimination factors D1 vary over 30–980 for the native and over 8–300 for the phosphorylated enzyme. From AMP formation stoichiometry and D1 values pretransfer proof-reading factors (II1) of 1.1–56 were calculated for the native enzyme, factors of 1.0–44 for the phosphorylated species. Post-transfer proof-reading factors (II2) were calculated from D values and AMP formation stoichiometry in acylation of tRNALys-C-C-A. Pretransfer proof-reading is the main correction step, posttransfer proof-reading is less effective or negligible (II2∼ 1–8). Initial discrimination factors (I), which are due to differences in Gibbs free energies of binding between lysine and noncognate substrates (ΔΔGI), were calculated from discrimination and proof-reading factors. In contrast to class I synthetases, for lysyl-tRNA synthetase only one initial discrimination step can be assumed and amino acid recognition is reduced to a three-step process instead of the four-step recognition observed for the class I synthetases. Plots of ΔΔGI values against accessible surface areas of amino acids show clearly that phosphorylation of the enzyme changes the structures of the amino acid binding sites. This is illustrated by a hypothetical ‘stopper model’ of these sites.

Discrimination factors (D) which are characteristic for discrimination between lysine and 19 naturally occurring non-cognate amino acids have been determined from kcat and Km values for native and phosphorylated lysyl-tRNA synthetases from yeast. Generally, both species of this class II aminoacyl-tRNA synthetase are considerably less specific than the class I synthetases specific for isoleucine, valine, tyrosine, and arginine. D values of the native enzyme are in the range 90-1700, D values of the phosphorylated species in the range 40-770. The phosphorylated enzyme acts faster and less accurately. In aminoacylation of tRNALys-C-C-A(2'NH2) discrimination factors D1 vary over 30-980 for the native and over 8-300 for the phosphorylated enzyme. From AMP formation stoichiometry and D1 values pretransfer proof-reading factors (II1) of 1.1-56 were calculated for for the native enzyme, factors of 1.0-44 for the phosphorylated species. Post-transfer proof-reading factors (II2) were calculated from D values and AMP formation stoichiometry in acylation of tRNALys-C-C-A. Pretransfer proof-reading is the main correction step, posttransfer proof-reading is less effective or negligible (II2 approximately 1-8). Initial discrimination factors (I), which are due to differences in Gibbs free energies of binding between lysine and noncognate substrates (delta delta GI), were calculated from discrimination and proof-reading factors. In contrast to class I synthetases, for lysyl-tRNA synthetase only one initial discrimination step can be assumed and amino acid recognition is reduced to a three-step process instead of the four-step recognition observed for the class I synthetases. Plots of delta delta GI values against accessible surface areas of amino acids show clearly that phosphorylation of the enzyme changes the structures of the amino acid binding sites. This is illustrated by a hypothetical 'stopper model' of these sites.

For discrimination between isoleucine and 19 other amino acids by isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600, discrimination factors D have been determined from kcat and Km values in amino-acylation of cognate tRNA(Ile)-C-C-A. Factors D are also products of initial discrimination factors I' and proof-reading factors II'; D = I' II'. Factors II' were calculated from AMP formation stoichiometries and factors I' as quotients of D and II'; I' = D/II'. II' is considered as a product of a pre- and post-transfer proof-reading factor (II' = II1II2), I' as a product of initial discrimination factors I1 and I2 which are due to two steps of initial discrimination. With the yeast enzyme the highest accuracy is achieved in discrimination between isoleucine and valine (D = 38,000); other D values in a high range (10,000-20,000) are observed for Gly, Ser, Thr, Leu and Met; the lowest factors D belong to Cys, Asp, Asn and Trp (300-700); the remaining amino acids are discriminated with medium D values (1000-10,000). Discrimination factors D observed for isoleucyl-tRNA synthetase from E. coli are on average 2-3 times higher than for the yeast enzyme. Highest values were found in discrimination against Gly, Ala and Val (60,000-72,000), the lowest values for Cys, Arg and Trp (600-3000); the other amino acids exhibit D values between 20,000 and 50,000. Initial discrimination factors can be related to hydrophobic interaction forces between the substrates and the enzyme; a hypothetical model of the amino acid binding site is discussed.

The order of substrate addition to tyrosyl-tRNA synthetase from baker's yeast was investigated by bisubstrate kinetics, product inhibition and inhibition by dead-end inhibitors. The kinetic patterns are consistent with a random bi-uni uni-bi ping-pong mechanism. Substrate specificity with regard to ATP analogs shows that the hydroxyl groups of the ribose moiety and the amino group in position 6 of the base are essential for recognition of ATP as substrate. Specificity with regard to amino acids is characterized by discrimination factors D which are calculated from kcat and Km values obtained in aminoacylation of tRNATyr-C-C-A. The lowest values are observed for Cys. Phe. Trp (D= 28000 – 40000), showing that, at the same amino acid concentrations, tyrosine is 28000 – 40000 times more often attached to tRNATyr-C-C-A than the noncognate amino acids. With Gly, Ala and Ser no misacylation could be detected (D > 500000); D values of the other amino acids are in the range of 100000—500000. Lower specificity is observed in aminoacylation of the modified substrate tRNATyr-C-C-A(3'NH2) (D1= 500—55000). From kinetic constants and AMP-formation stoichiometry observed in aminoacylation of this tRNA species, as well as in acylating tRNATyr-C-C-A hydrolytic proof-reading factors could be calculated for a pretransfer (II1) and a post-transfer (II2) proof-reading step. The observed values of II1= 12 – 280 show that pretransfer proof-reading is the main correction step whereas post-transfer proof-reading is marginal for most amino acids (II2= 1–2). Initial discrimination factors caused by differences in Gibbs free energies of binding between tyrosine and noncognate amino acids are calculated from discrimination and proof-reading factors. Assuming a two-step binding process, two factors (I1 and I2) are determined which can be related to hydrophobic interaction forces. The tyrosine side chain is bound by hydrophobic forces and hydrogen bonds formed by its hydroxyl group. A hypothetical model of the amino acid binding site is discussed and compared with results of X-ray analysis of the enzyme from Bacillus stearothermophilus.

The localization of the binding sites of the different ligands on the constitutive subunits of yeast phenylalanyl-tRNA synthetase was undertaken using a large variety of affinity and photoaffinity labelling techniques. The RNAPhe was cross-linked to the enzyme by non-specific ultraviolet irradiation at 248 nm, specific irradiation in the wye base absorption band (315 nm), irradiation at 335 nm, in the absorption band of 4-thiouridine (S4U) residues introduced in the tRNA molecule, or by Schiff's base formation between periodate-oxidized tRNAPhe (tRNAPheox) and the protein. ATP was specifically incorporated in its binding site upon photosensitized irradiation. The amino acid could be linked to the enzyme upon ultraviolet irradiation, either in the free state, engaged in the adenylate or bound to the tRNA. The tRNA, the ATP molecule and the amino acid linked to the tRNA were found to interact exclusively with the beta subunit (Mr 63000). The phenylalanine residue, either free or joined to the adenylate, could be cross-linked with equal efficiency to eigher type of subunit, suggesting that the amino acid binding site is located in a contact area between the two subunits. The Schiff's base formation between tRNAPheox and the enzyme shows the existence of a lysyl group close to the binding site for the 3'-terminal adenosine of tRNA. This result was confirmed by the study of the inhibition of yeast phenylalanyl-tRNA synthetase with pyridoxal phosphate and the 2',3'-dialdehyde derivative of ATP, oATP.

Determination of the molecular weight of valyl and isoleucyl tRNA synthetases under denaturing conditions leads to the conclusion that both enzymes are made of a single polypeptide chain of a molecular weight of 110000 approximately. The presence of one amino acid binding site per chain was determined by equilibrium dialysis.

The kinetics of phenylalanyl-tRNA and seryl-tRNA formation were investigated with tRNAs and aminoacyl-tRNA synthetases from yeast. Phenylalanyl-tRNA synthetase yielded linear Lineweaver-Burk plots with tRNAphe, phenylalanine, and 1,N6-ethenoadenosine triphosphate (ɛATP) as variable substrates. According to equilibrium dialysis in the absence or presence of phenylalaninyl adenosine 5′-phosphate, phenylalanyl-tRNA synthetase possesses one binding site for phenylalanine. For ATP as variable substrate, the deviation from linearity in the Lineweaver-Burk plot, observed by other investigators, was confirmed. The slope of the curve indicates the presence of more than two ATP binding sites. Seryl-tRNA synthetase yielded a linear Lineweaver-Burk plot only with ɛATP as variable substrate. The Lineweaver-Burk plots for serine and tRNASer were non-linear; the interpretation we favor involves positive cooperativity between amino acid binding sites and between tRNA binding sites. Hill plots of the kinetic data showed that the enzyme possesses at least two binding sites for each of these substrates. The kinetic data for ATP could be interpreted as showing more than two binding sites with negative and positive cooperativity in binding of successive ATP molecules. The aminoalkyl adenylates, phenylalaninyl adenosine 5′-phosphate and serinyl adenosine 5′-phosphate, competitively inhibited the aminoacylation reaction with respect to amino acid. ɛATP functions in place of ATP in phenylalanyl-tRNA and seryl-tRNA formation although with rather different kinetic properties. Modified tRNAphe and tRNASer, in which the 3′-terminal adenosine was replaced by ethenoadenosine, were prepared by a C-C-A transferase-catalyzed reaction of ɛATP. These modified tRNAs show kinetic properties very similar to those of the unmodified tRNAs and can therefore be used, in place of the unmodified tRNAs, as fluorescent probes in synthetase-tRNA interaction studies. The kinetic behavior of phenylalanyl-tRNA synthetase appears to be much simpler than that of seryl-tRNA synthetase, despite the fact that the former enzyme is twice as big and contains twice as many subunits as the latter one. The comparative simplicity of the one enzyme relative to the other correlates with previous results on interactions with substrates, which were obtained by fluorescence measurements and nuclease protection studies.