| Charcot-Marie-Tooth disorder (CMT) is the most common inherited peripheral neuropathy, afflicting one in every 2500 Americans (1). Dominant-intermediate Charcot-Marie-Tooth disorder type C (DI-CMTC) is due to three mutations in the gene encoding tyrosyl-tRNA synthetase (TyrRS) (2). These changes in tyrosyl-tRNA synthetase are G41R, E196K, and Delta(l 53-156). We tested the hypothesis that DI-CMTC is due to a defect in the ability to catalyze the transfer of tyrosine to tRNATyr and found that two of the variants, G41R and Delta(153-156), have severe catalytic defects in the formation of the TyrRS·Tyr-AMP intermediate. The E196K variant activates and transfers tyrosine to tRNATyr similar to the wild-type enzyme. To test the hypothesis that the E196K variant is less stable than the wild type enzyme, limited proteolysis studies were performed. All three of tyrosyl-tRNA synthetase variants have similar stabilities as the wild type enzyme. We examined whether the E196K variant is less specific for noncognate amino acids than the wild type enzyme. Little difference is observed between the abilities of the E196K variant and wild type enzyme to discriminate between cognate and noncognate amino acids. The E196K variant was found to be much less specific than the wild type enzyme with respect to tRNA recognition, The E196K variant of is able to efficiently aminoacylate bacterial tRNATyr. We propose that E196 acts as an anti-determinant, preventing human tyrosyl-tRNA synthetase from recognizing tRNAs with a G1:C72 base pair. We find that the E196K has a 100-fold higher specificity constant for the G1·C72 human tRNA Tyr than the wild type enzyme. These data support the hypothesis that three of the variants of tyrosyl-tRNA synthetase that give rise to DI-CMTC result in a defect in protein synthesis. |
