Modification of l-Isoleucyl-tRNA Synthetase with l-Isoleucyl-Bromomethyl Ketone. The Effect on 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, 419-4261, 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-tRNA"" 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 cysteinesulfhydryl group during catalysis.

Interaction of pseudomonic acid A with Escherichia coli B isoleucyl-tRNA synthetase

“…Previous work has shown that sodium pseudomonate causes arrest of protein synthesis in prokaryotic organisms both in vivo and in vitro by specifically interfering with the function of isoleucyl-tRNA synthetase (Hughes & Mellows, 1978a,b (Rainey et aL, 1977). t Km (literature) 4-6juM (Cole & Schimmel, 1970;Fersht & Kaethner, 1976;Helene et al, 1971;Loftfield & Eigner, 1965;Rainey et al, 1977).…”

“…t Km (literature) 4-6juM (Cole & Schimmel, 1970;Fersht & Kaethner, 1976;Helene et al, 1971;Loftfield & Eigner, 1965;Rainey et al, 1977). (a) the overall aminoacylation of tRNAIIe and the pyrophosphate-exchange reaction were competitively inhibited by sodium pseudomonate in the concentration range 0-5OnM, with respect to isoleucine;…”

Aminoacyl-tRNA Synthetases: General Features and Recognition of Transfer RNAs

“…Studies on the mechanism of these enzymes are proceeding at a fa st pace (101,(324)(325)(326)(327)(328)(329). Both kinetic and thermodynamic analyses are being done.…”

Catalytic mechanism of phenylalanyl-tRNA synthetase of Escherichia coli K10. Conformational change and tRNAPhe phenylalanylation are concerted

“…Enzyme-phenylalanyl adenylate was prepared in situ by incubation of enzyme, ATP, Phe, and inorganic pyrophosphatase (1-10 jig/mL) in one syringe of the stopped-flow apparatus. Enzyme-phenylalanyl adenylate complex free of reactants was prepared by passing the reaction mixture of Sephadex G50 coarse as described (Rainey et al, 1977). In one experiment, in situ prepared enzyme-adenylate was separated kinetically from reactive Phe by including L-phenylalaninol in the tRNA solution.…”