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 pre-steady-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 E.Tyr.ATP and E.[Tyr-ATP] complexes by 2.3 and 4.3 kcal/mol, respectively. Potassium also appears to stabilize the asymmetric conformation of the human tyrosyl-tRNA 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.