title: Modification of L-isoleucyl-tRNA synthetase with L-isoleucyl-bromomethyl ketone. The effect of the catalytic steps.
abstract: The rapidly reacting cysteine-sulfhydryl group of L-isoleucyl-tRNA synthetase has been specifically alkylated with L-isoleucyl-bromomethyl ketone [Rainey, P., Holler, E. & Kula, M.-R. (1976) Eur. J. Biochem. 63, 419-426]. We have now investigated the catalytic and substrate binding properties of the modified protein by radioactive and fluorescence techniques. The rate constants for the transfer of AMP and isoleucine from the protein - adenylate complex to form ATP or Ile-tRNAIle were only 3% of those for native enzyme, whereas the rate constant for the formation of adenylate was essentially unchanged. The tendency to form synthetase - substrate complexes remained almost unchanged with the exception of L-isoleucine which exhibited a 20-fold reduction. Similarly, complex formation of L-isoleucinol together with its synergistic coupling to complex formation of ATP was partially inhibited. The results rule out the essential participation of the rapidly alkylatable cysteine-sulfhydryl group during catalysis.
mesh_terms: D000255:Adenosine Triphosphate; D000345:Affinity Labels; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology; D007532:Isoleucine / Q000031:analogs & derivatives*; D007533:Isoleucine-tRNA Ligase / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D012343:RNA, Transfer
chemical_list: D000345:Affinity Labels; D007532:Isoleucine; D000255:Adenosine Triphosphate; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; D007533:Isoleucine-tRNA Ligase; D003545:Cysteine