Discrimination of Cognate and Non-Cognate Substrates at the Active Site of Class I Lysyl-tRNA Synthetase
The aminoacyl-tRNA synthetases are divided into two unrelated structural classes, with lysyl-tRNA synthetase (LysRS) being the only enzyme represented in both classes. On the basis of the structure of l-lysine complexed with Pyrococcus horikoshii class I LysRS (LysRS1) and homology to glutamyl-tRNA synthetase (GluRS), residues implicated in amino acid recognition and noncognate substrate discrimination were systematically replaced in Borrelia burgdorferi LysRS1. The catalytic efficiency of steady-state aminoacylation (kcat/KM) with lysine by LysRS1 variants fell by 1−4 orders of magnitude compared to that of the wild type. Disruption of putative hydrogen bonding interactions through replacement of G29, T31, and Y269 caused up to 1500-fold reductions in kcat/KM, similar to changes previously observed for comparable variants of class II LysRS (LysRS2). Replacements of W220 and H242, both of which are implicated in hydrophobic interactions with the side chain of lysine, resulted in more dramatic changes with up to 40000-fold reductions in kcat/KM observed. This indicates that the more compact LysRS1 active site employs both electrostatic and hydrophobic interactions during lysine discrimination, explaining the ability of LysRS1 to discriminate against noncognate substrates accepted by LysRS2. Several of the LysRS1 variants were found to be more specific than the wild type with respect to noncognate amino acid recognition but less efficient in cognate aminoacylation. This indicates that LysRS1 compromises between efficient catalysis and substrate discrimination, in contrast to LysRS2 which is considerably more effective in catalysis but is less specific than its class I counterpart.
Wang, S., Prætorius-Ibba, M., Ataide, S., Roy, H. and Ibba, M. (2006). Discrimination of cognate and non-cognate substrates at the active site of class I lysyl-tRNA synthetase. Biochemistry 45, 3646-3652. https://doi.org/10.1021/bi0523005
American Chemical Society