Active site of an aminoacyl-tRNA synthetase dissected by energy-transfer-dependent fluorescence.
Aminoacyl-tRNA synthetases establish the rules of the genetic code by catalyzing attachment of amino acids to specific transfer RNAs (tRNAs) that bear the anticodon triplets of the code. Each of the 20 amino acids has its own distinct aminoacyl-tRNA synthetase. Here we use energy-transfer-dependent fluorescence from the nucleotide probe N-methylanthraniloyl dATP (mdATP) to investigate the active site of a specific aminoacyl-tRNA synthetase. Interaction of the enzyme with the cognate amino acid and formation of the aminoacyl adenylate intermediate were detected. In addition to providing a convenient tool to characterize enzymatic parameters, the probe allowed investigation of the role of conserved residues within the active site. Specifically, a residue that is critical for binding could be distinguished from one that is important for the transition state of adenylate formation. Amino acid binding and adenylate synthesis by two other aminoacyl-tRNA synthetases was also investigated with mdATP. Thus, a key step in the synthesis of aminoacyl-tRNA can in general be dissected with this probe.
Nomanbhoy
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The Skaggs Institute for Chemical Biology, The Scripps Research Institute, Beckman Center, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Schimmel
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D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes; D007533:Isoleucine-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D017354:Point Mutation; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase