Expression and characterization of the human mitochondrial leucyl-tRNA synthetase.
A cDNA clone encoding the human mitochondrial leucyl-tRNA synthetase (mtLeuRS) has been identified from the EST databases. Analysis of the protein encoded by this cDNA indicates that the protein is 903 amino acids in length and contains a mitochondrial signal sequence that is predicted to encompass the first 21 amino acids. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl-tRNA synthetases and regions of high homology to other mitochondrial and bacterial LeuRS proteins. The mature form of this protein has been cloned and expressed in Escherichia coli. Gel filtration indicates that human mtLeuRS is active in a monomeric state, with an apparent molecular mass of 101 kDa. The human mtLeuRS is capable of aminoacylating E. coli tRNA(Leu). Its activity is inhibited at high levels of either monovalent or divalent cations. K(M) and k(cat) values for ATP:PP(i) exchange and for the aminoacylation reaction have been determined.
Bullard
J M
JM
Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599-3290, USA.
Cai
Y C
YC
Spremulli
L L
LL
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D005075:Biological Evolution; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000737:chemistry / Q000145:classification / Q000235:genetics*; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D012343:RNA, Transfer / Q000737:chemistry; D016415:Sequence Alignment; D017385:Sequence Homology; D013045:Species Specificity
D018076:DNA, Complementary; D012333:RNA, Messenger; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D007935:Leucine-tRNA Ligase