| Due to their essential role in the process of protein synthesis in all forms of life, aminoacyl-tRNA synthetases are excellent targets for the design of antibiotics. The goal of the research presented here is to determine whether mechanistic differences exist between the human and bacterial tyrosyl-tRNA synthetase that could potentially be exploited in the design of antibacterial agents. The catalytic mechanism of tyrosine activation by the human tyrosyl-tRNA synthetase was characterized and compared to that of the B. stearothermophilus enzyme. In the B. stearothermophilus tyrosyl-tRNA synthetase, stabilization of the transition state for tyrosine activation is largely due to the formation of interactions with the ATP substrate. Four of the residues that have been shown to make these interactions in the B. stearothermophilus tyrosyl-tRNA synthetase are not conserved in the human enzyme (Cys 35, His 48, Thr 51, and Lys 233). Cys 35, His 48, and Thr, 51 interact with the ribose ring of ATP, while Lys 233 interacts with the pyrophosphate moiety. Pre-steady state kinetic analyses of the human tyrosyl-tRNA synthetase indicates that despite the absence of conservation of these residues in the human tyrosyl-tRNA synthetase, the stabilities of the transition states for tyrosine activation are virtually identical for the B. stearothermophilus and human tyrosyl-tRNA synthetases. These results suggest that the human tyrosyl-tRNA synthetase somehow compensates for the absence of these residues. Kinetic analyses revealed that, unlike the B. stearothermophilus tyrosyl-tRNA synthetase, the human enzyme requires potassium for catalysis. Specifically, potassium stabilizes the transition state complex of the reaction through interactions with the pyrophosphate moiety of ATP. These results are consistent with the hypothesis that potassium functionally compensates for the absence of conservation of the most highly conserved residue among Class I aminoacyl-tRNA synthetases, the second lysine in the KMSKS signature sequence. These observations are consistent with a mechanistic difference existing between the human and bacterial tyrosyl-tRNA synthetases that could potentially be exploited in the design of novel inhibitors that selectively inhibit the bacterial enzyme. |
