Characterization of the human mitochondrial methionyl-tRNA synthetase

Human mitochondrial methionyl-tRNA synthetase (human mtMetRS) has been identified from the human EST database. The cDNA encodes a 593 amino acid protein with an 18 amino acid mitochondrial import signal sequence. Sequence analysis indicates that this protein contains the consensus motifs characteristic of a class I aminoacyl-tRNA synthetase but lacks the Zn ²⁺ binding motif and C-terminal dimerization region found in MetRSs from various organisms. The mature form of human mtMetRS has been cloned and expressed in Escherichia coli. Gel filtration experiments indicate that this protein functions as a monomer with an apparent molecular mass of 67 kDa. The kinetic parameters for activation of methionine have been determined for the purified enzyme. The K_M and k_cat for aminoacylation of E. coli initiator tRNA_t^Met are reported. The kinetics of aminoacylation of an in vitro transcript of human mitochondrial tRNA ^Met (mtRNA^Met) have been determined. To address the effects of the modification of mtRNA on recognition of the mitochondrial tRNA by human mtMetRS, the kinetics of aminoacylation of native bovine mtRNA ^Met and of an in vitro transcript of the bovine mtRNA^Met have also been investigated.