Potassium Functionally Replaces the Second Lysine of the KMSKS Signature Sequence in Human Tyrosyl-tRNA Synthetase

Abstract

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...

Comparison of the Catalytic Roles Played by the KMSKS Motif in the Human and Bacillus stearothermophilus Tyrosyl-tRNA Synthetases

“…In addition, quantitatively comparing the kinetics for alanine variants at equivalent positions in the human and B. stearothermophilus tyrosyl-tRNA synthetases indicates that although the KMSKS motifs play similar roles in the two enzymes, the extent to which the KMSKS motif catalyzes the formation of tyrosyl-adenylate is significantly larger in the B. stearothermophilus enzyme than it is in the human enzyme. This is consistent with the observation that potassium appears to compensate for the absence of the second lysine in the KMSKS motif of the human tyrosyl-tRNA synthetase (19). The observation that the KMSKS motif plays a larger role in the catalytic mechanism of the B. stearothermophilus tyrosyl-tRNA synthetase suggests that inhibitors that interact with this mo- tif will have higher affinities for bacterial tyrosyl-tRNA synthetases than for their eukaryotic homologs.…”

“…Previous investigations indicate that the loss of catalytic function by the KMSSS sequence is compensated for by the involvement of potassium in the catalytic mechanism of human tyrosyl-tRNA synthetase (19). Specifically, potassium has been shown to functionally compensate for the absence of the second lysine in the KMSSS sequence (19).…”

“…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 how human tyrosyl-tRNA synthetase compensates for the absence of the second lysine in the KMSKS signature sequence. Recently, it was shown that in the human tyrosyltRNA synthetase, potassium stabilizes the transition state for tyrosine activation by interacting with the pyrophosphate moiety of ATP (19). Based on these observations, it was concluded that in human tyrosyl-tRNA synthetase, potassium functionally replaces the second lysine in the KMSKS signature sequence.…”

Structural Snapshots of the KMSKS Loop Rearrangement for Amino Acid Activation by Bacterial Tyrosyl-tRNA Synthetase

“…The KMSKS loops of these enzymes require potassium ions for catalysis, 16 and are somewhat different from those of the bacterial TyrRSs. 17 Further analyses of the archaeal/ eukaryal TyrRSs will reveal the detailed role of the KMSKS loop.…”

“…Structural analyses and some biochemical analyses revealed that the KMSKS loops, as well as the tRNA-recognition sites, differ between the bacterial and archaeal/ eukaryal TyrRSs. [15][16][17][18] An abundance of biochemical information is available for the bacterial TyrRSs, [6][7][8][9][10] and the L-tyrosine-bound, 4 the ATP and L-tyrosine analogue-bound 13 and the Tyr-AMP-bound 5 structures are now available. In contrast, very little data exist regarding the catalytic mechanism of the KMSKS loop in the archaeal/eukaryal TyrRSs.…”