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 KM and kcat for aminoacylation of E. coli initiator tRNAtMet are reported. The kinetics of aminoacylation of an in vitro transcript of human mitochondrial tRNA Met (mtRNAMet) 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 mtRNAMet have also been investigated.
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