Biochemical properties of class I lysyl-tRNA synthetase

by 1978- Levengood, Jeffrey David

The family of aminoacyl-tRNA synthetases (aaRSs) performs the essential cellular function of charging tRNA molecules with their cognate amino acids. This enzyme family can be divided into two unrelated classes with each evolving from different origins. Lysyl-tRNA synthetase (LysRS) is the only synthetase known to have a form in each class. The class I form (LysRS1) is found in most archaea and a few bacteria while the class II form (LysRS2) is found in all eucarya, most bacteria, and a few archaea. Steady-state kinetics were used to study the mechanisms which LysRS1 employs to recognize its substrates. The binding of both amino acid and tRNA were examined. The binding of lysine was analyzed by studying the ability of several lysine analogues to inhibit the aminoacylation reaction. It was found that the R-group plays a critical role in discrimination rather than the functional groups at the ?-carbon. Within the R-group, the size of the chain was a very important point for discrimination. The binding of tRNALys was examined using both LysRS, mutated based on the modeled tRNALys bound to LysRS1, and tRNALys with mutations in the anticodon. ii Analysis of the bound tRNALys revealed that LysRS is able to specifically recognize the bases at anticodon positions 35 and 36, but not position 34. The experiments with variant enzymes in relation to the model revealed LysRS1 likely uses a variety of bonds to recognize the anticodon. Aromatic residues form non-specific stacking interactions with the bases, charged and polar residues form specific hydrogen bonds with the bases, and other charged residues form non-specific electrostatic interactions with the phosphate backbone of the anticodon stem. There was also evidence that single point mutations in amino acid residues binding the anticodon could have major effects on anticodon recognition and the ability to rescue tRNA mutations. Comparison of the mechanisms for substrate recognition between LysRS1 and LysRS2 revealed differences in the recognition of substrates that could be the cause for the divergence of the two enzymes. The two LysRSs were shown to have different patterns of inhibition to naturally occurring lysine analogues that had consequences for growth in vivo. With the tRNALys anticodon binding, it was found anticodon binding is much more important for binding with LysRS2 than LysRS1. This could have had consequences with the divergence of other synthetases that must recognize similar anticodons. The presence of LysRS1 may have allowed these other synthetases to put more emphasis on the anticodon in recognition of their cognate tRNA. iii