Although the active site residues in the Bacillus stearothermophilus and human tyrosyl-tRNA synthetases are largely conserved, several differences exist between the two enzymes. In particular, three amino acids that stabilize the transition state for the activation of tyrosine in B. stearothermophilus tyrosyl-tRNA synthetase (Cys-35, His-48, and Lys-233) are not present in the human enzyme. This raises the question of whether the activation energy for the tyrosine activation step is higher for the human tyrosyl-tRNA synthetase than for the B. stearothermophilus enzyme. In this paper, we demonstrate that intrinsic fluorescence changes can be used to monitor the pre-steady state kinetics of human tyrosyl-tRNA synthetase. In contrast to the B. stearothermophilus enzyme, catalysis of the tyrosine activation step is potassium-dependent in the human tyrosyltRNA synthetase. Specifically, potassium increases the forward rate constant for tyrosine activation 260-fold in the human tyrosyl-tRNA synthetase. Comparison of the forward rate constants for catalysis of tyrosine activation by the human and B. stearothermophilus enzymes indicates that despite differences in their active sites and the potassium requirement of the human enzyme, the activation energies for tyrosine activation are identical for the two enzymes. The results of these investigations suggest that differences exist between the active sites of the bacterial and human tyrosyl-tRNA synthetases that could be exploited to design antimicrobials that target the bacterial enzyme.Aminoacyl-tRNA synthetases catalyze the transfer of amino acids to tRNA by a two-step reaction. In the first step, the amino acid substrate reacts with MgATP to form the enzymebound aminoacyl-adenylate intermediate. In the second step of the reaction, the aminoacyl moiety is transferred from the aminoacyl-adenylate intermediate to the 3Ј end of tRNA. There are two distinct classes of aminoacyl-tRNA synthetases. The Class I aminoacyl-tRNA synthetase family, of which tyrosyltRNA synthetase is a member, is characterized by an aminoterminal Rossmann-fold and conserved "HIGH" and "KMSKS" signature sequences. In tyrosyl-tRNA synthetase, the conserved HIGH and KMSKS signature sequences stabilize the transition state for the first step of the reaction (1-8).Bacillus stearothermophilus tyrosyl-tRNA synthetase is a homodimeric enzyme that displays "half-of-the-sites" reactivity with respect to tyrosine binding and tyrosyl-adenylate formation (9 -11). Site-directed mutagenesis and pre-steady state kinetic analyses have been used to identify 18 active site amino acids that stabilize the transition state for tyrosine activation in the B. stearothermophilus enzyme (reviewed in Refs. 12 and 13). Four of these amino acids are absent in the human tyrosyltRNA synthetase. In the B. stearothermophilus enzyme, replacement of three of these amino acids, Cys-35, His-48, and Lys-233, destabilizes the transition state for tyrosine activation by 1.2, 1.2, and 3.0 kcal/mol, respectively (1-6, 14). His-48 and Lys-233...

Unlike their bacterial homologues, a number of eukaryotic tyrosyl-tRNA synthetases require potassium to catalyze the aminoacylation reaction. In addition, the second lysine in the class I-specific KMSKS signature motif is absent from all known eukaryotic tyrosyl-tRNA synthetase sequences, except those of higher plants. This lysine, which is the most highly conserved residue in the class I aminoacyl-tRNA synthetase family, has been shown to interact with the pyrophosphate moiety of the ATP substrate in the Bacillus stearothermophilus tyrosyl-tRNA synthetase. Equilibrium dialysis and presteady-state kinetic analyses were used to determine the role that potassium plays in the tyrosine activation reaction in the human tyrosyl-tRNA synthetase and whether it can be replaced by any of the other alkali metals. Kinetic analyses indicate that potassium interacts with the pyrophosphate moiety of ATP, stabilizing the E⅐Tyr⅐ATP and E⅐[Tyr-ATP] ‡ complexes by 2.3 and 4.3 kcal/mol, respectively. Potassium also appears to stabilize the asymmetric conformation of the human tyrosyltRNA synthetase dimer by 0.7 kcal/mol. Rubidium is the only other alkali metal that can replace potassium in catalyzing tyrosine activation, although the forward rate constant is half of that observed when potassium is present. The above results are consistent with the hypothesis that potassium functionally replaces the second lysine in the KMSKS signature sequence. Possible implications of these results with respect to the design of antibiotics that target bacterial aminoacyl-tRNA synthetases are discussed.Aminoacyl-tRNA synthetases have gained attention recently as potential targets for antibiotics (1-10). Identifying differences in the catalytic mechanisms of bacterial and human aminoacyl-tRNA synthetases will facilitate the development of antibiotics that selectively target the bacterial aminoacyltRNA synthetases. We are currently investigating the catalytic mechanisms of the human and Bacillus stearothermophilus tyrosyl-tRNA synthetases to elucidate the differences between these two enzymes.Tyrosyl-tRNA synthetase catalyzes the attachment of tyrosine to tyrosine tRNA (tRNA Tyr ) 1 by an ATP-dependent twostep reaction mechanism. In the first step, tyrosine is activated by MgATP to form an enzyme-bound tyrosyl-adenylate intermediate. The second step consists of the transfer of tyrosine to the 3Ј end of tRNA Tyr .TyrRS ϩ Tyr ϩ MgATP^TyrRS ⅐ Tyr-AMP ϩ PP i (Eq. 1)Tyrosyl-tRNA synthetase is a homodimer that displays "halfof-the-sites" reactivity, with only one tyrosyl-adenylate molecule formed per dimer (11-13). Although most of the active site amino acids are conserved between the human and B. stearothermophilus tyrosyl-tRNA synthetases, several differences exist between the two enzymes, including the inability of the human and bacterial tyrosyl-tRNA synthetases to aminoacylate each other's tRNA Tyr (14,15). In addition, sequence analyses indicate that the human tyrosyl-tRNA synthetase is Ͻ16% identical to the B. stearothermophilus enzyme and that four...

As the number of prescriptions, over-the-counter medications and drugs of abuse continue to increase, forensic laboratories are faced with the challenge of developing more comprehensive screening methods in order to detect them in whole blood samples. Another challenge faced by forensic laboratories is detecting and identifying novel synthetic compounds as they emerge and change. Traditional drug screening methods include enzyme immunoassay (EIA) and either gas or liquid chromatography paired with mass spectrometry (GC–MS or LC–MS-MS, respectively). While these methods are good, they have their disadvantages. For example, EIA requires special reagents for each drug class, GC–MS requires extensive sample preparation, and LC–MS-MS only detects drugs on a known inclusion lists of compounds of interest. Described below is the development of a robust and comprehensive screening method for drugs in whole blood samples that eliminates the aforementioned disadvantages of the traditional methods. Using a Q Exactive Focus ™ liquid chromatography-high resolution-accurate mass spectrometer (LC–HRMS-MS), a method was developed that is capable of detecting approximately 200 drugs at a concentration of 2 μg/L for most analytes. This method also employs a more automated data processing feature which reduces processing time. Finally, it has the added benefit of retroactive data analysis, which allows it to be used for unknown drug analysis as well. Used as an initial screening method, the comprehensive drug screen using LC–HRMS-MS has the potential to take on two of the most important challenges faced by forensic laboratories today.

The Class I aminoacyl-tRNA synthetases are characterized by two signature sequence motifs, "HIGH" and "KMSKS." In Bacillus stearothermophilus tyrosyl-tRNA synthetase, the KMSKS motif ( 230 KFGKT 234 ) has been shown to stabilize the transition state for tyrosine activation through interactions with the pyrophosphate moiety of ATP. In most eukaryotic tyrosyl-tRNA synthetases, the second lysine in the KMSKS motif is replaced by a serine or an alanine residue. Recent kinetic studies indicate that potassium functionally compensates for the absence of the second lysine in the human tyrosyl-tRNA synthetase ( 222 KKSSS 226 ). In this paper, site-directed mutagenesis and pre-steady state kinetics are used to determine the roles that serines 224, 225, and 226 play in catalysis of the tyrosine activation reaction. In addition, the catalytic role played by a downstream lysine conserved in eukaryotic tyrosyl-tRNA synthetases, Lys-231, is investigated. Replacing Ser-224 and Ser-226 with alanine decreases the forward rate constant 7.5-and 60-fold, respectively. In contrast, replacing either Ser-225 or Lys-231 with alanine has no effect on the catalytic activity of the enzyme. These results are consistent with the hypothesis that the KMSSS sequence in human tyrosyl-tRNA synthetase stabilizes the transition state for the tyrosine activation reaction by interacting with the pyrophosphate moiety of ATP. In addition, although they play similar roles in catalysis, the overall contribution of the KMSKS motif to catalysis appears to be significantly less in human tyrosyl-tRNA synthetase than it is in the B. stearothermophilus enzyme.Aminoacyl-tRNA synthetases (AARS) catalyze the attachment of amino acids (AA) to their cognate tRNA AA by an ATPdependent two-step reaction mechanism. In the first step (Equation 1), the amino acid is activated by MgATP to form an enzyme-bound aminoacyl-adenylate intermediate. The second step (Equation 2) consists of the transfer of the amino acid to the 3Ј end of its cognate tRNA AA .The Class I aminoacyl-tRNA synthetase family, of which tyrosyl-tRNA synthetase is a member, is characterized by the presence of an amino-terminal Rossmann-fold catalytic domain and conserved HIGH and KMSKS signature sequences (1-9). The KMSKS signature sequence in the Bacillus stearothermophilus tyrosyl-tRNA synthetase ( 230 KFGKT 234 ) participates in catalysis of the tyrosine activation reaction (10 -15). Specifically, Lys-230, Lys-233, and Thr-234 stabilize the transition state by interacting with the pyrophosphate moiety of the ATP substrate (11-15). In the human tyrosyl-tRNA synthetase Gly-232, Lys-233, and Thr-234 are replaced with serine residues ( 222 KMSSS 226 ) (16). The absence of a second lysine in the KMSSS sequence in human tyrosyl-tRNA synthetase, which is the most highly conserved amino acid in the Class I aminoacyltRNA synthetase family (17), and the observation that the catalytic efficiency of human tyrosyl-tRNA synthetase is similar to that of the B. stearothermophilus enzyme (18) raises the question of ho...

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