Non-standard amino acid recognition by Escherichia coli leucyl-tRNA synthetase.
Recombinant E. coli leucyl-tRNA synthetase was screened for amino acid-dependent pyrophosphate exchange activity using noncognate aliphatic amino acids including norvaline, homocysteine, norleucine, methionine, and homoserine. [32P]-labeled reaction products were separated by thin layer chromatography using a novel solvent system and then quantified by phosphorimaging. Norvaline which differs from leucine by only one methyl group stimulated pyrophosphate exchange activity as did both homocysteine and norleucine to a lesser extent. The KM parameters for leucine and norvaline were measured to be 10 micromoles and 1.5 mM, respectively. Experiments are in progress to determine if norvaline is transferred to tRNA(Leu) and/or edited by a pre- or post-transfer mechanism.
Martinis
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Department of Biochemical and Biophysical Sciences, University of Houston, TX 77204-5934, USA.
Fox
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D002855:Chromatography, Thin Layer; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006710:Homocysteine / Q000378:metabolism; D006868:Hydrolysis; D007935:Leucine-tRNA Ligase / Q000378:metabolism*; D012329:RNA, Bacterial; D012356:RNA, Transfer, Leu / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D014633:Valine / Q000031:analogs & derivatives* / Q000378:metabolism
D000596:Amino Acids; D012329:RNA, Bacterial; D012356:RNA, Transfer, Leu; D011993:Recombinant Fusion Proteins; D006710:Homocysteine; D000255:Adenosine Triphosphate; C005313:norvaline; D007935:Leucine-tRNA Ligase; D014633:Valine