Search

The binding of nine aminoalkyl adenylates to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 was measured and compared with the binding of the cognate amino acids. It was found that they bind rather tightly to the enzyme, the Kd's ranging from 3.1.10(-4) M with glycinol-AMP ester to 3.7.10(-9) M with L-isoleucinol-AMP ester. The binding is not affected by magnesium. It is shown that the free energies of binding of the esters can be calculated adding a constant contribution of the AMP-moiety of about - 4.1 (- 17) kcal/mole (kJ/mole) to the free energies of binding of the cognate amino acids, which we have reported earlier (19, 25, 26).

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.

The partition behavior of isoleucyl-tRNA synthetase, leucyl-tRNA synthetase and tRNA in aqueous two-phase systems composed of the polymers poly(ethyleneglycol) and dextran was investigated. From the results of this investigation a two-phase system could be derived which can be employed for the study of the interactions between synthetases and their cognate tRNAs by equilibrium partition. These measurements show that in each case one molecule of cognate tRNA is bound per molecule of enzyme. The binding constants were in the range 1-5micronM-1. It could be demonstrated that equilibrium partition is a useful method for the study of interactions between macromolecules.

The binding of 10 isomeric alpha-amino-heptanoic acids, of two isomeric alpha-amino-octanoic acids, of isoleucinol and valinol, and of various alpha-hydroxy acids to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 has been investigated by an ultracentrifuge method. It was found that the enzyme requires a primary amino group together with a not-too-small side chain as prerequisites for ligand recognition. Though the enzyme is absolutely specific for the L isomers, it is fairly tolerant against replacement of the carboxyl group of the natural substrate by more or less hydrophobic substituents. These findings can be explained in terms of Ogston's three-point-attachment model, if it is additionally assumed that there is no further space available in the binding region normally occupied by the alpha-hydrogen atom to accept other substituents which are as bulky as the carboxyl moiety. Similarly, the architecture of the binding region of the aliphatic side chain is discussed. Our measurements show that the free energy of binding strongly depends on the size and the structure of the remainder of the molecule. None of the isoleucine analogues employed is bound as tightly as the natural substrate itself, but there is a clear preference for side chains branched at the beta-carbon atom. The functioning of the side-chain recognition site is best understood by imaging a two-finger glove, of which one finger is tailored to a methyl and the other to an ethyl group. Both these fingers, together with the binding region for the glycine moiety and a steric barrier against a fourth substituent bulkier than hydrogen, are responsible for a high steric specificity towards the one side chain over its Cbeta epimer.

Aminoalcohol-AMP esters, structurally related to the assumed intermediates of the amino acid activation reaction, behave as competitive inhibitors both with respect to the amino acid and ATP, when tested in the ATP-(32P) PPi-exchange or the tRNA-charging reaction. However, closer investigation of the binding of norvalinyl adenylate to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 by an equilibrium method shows that only the amino acid is a true competitor, while ATP cannot displace the ester from binding. Pyrophosphate enhances the stability of the ester-enzyme complex whereas tRNA is without detectable influence.

L-Isoleucine: tRNA ligase from Escherichia coli could be irreversibly inactivated by L-isoleucyl-bromomethyl ketone but not by L-isoleucyl-chromethyl ketone. The inactivation rate exhibited a saturation concentration dependence typical for an affinity reagent. L-Isoleucine provided 100% protection against inactivation at saturating concentration, whereas ATP, AMP, and pyrophosphate offered partial protection and tRNAIle, no protection. The ligase was labelled in preparative scale with L-[14C]isoleucyl-bromethyl ketone. The molar ration of label incorporated to enzyme inactivated was close to unity. The protein was subsequently subjected to tryptic digestion and the radioactive peptide isolated and identified. The labelled amino acid proved to be the same cysteine previously reported as being labelled with N-[14C]ethylmaleimide [Kula, M.-R. (1974) FEBS Lett. 46, 130-133].

The association enthalpies, delta Ha, involved in the reactions between L-isoleucine:tRNA ligase (AMP-forming) from Escherichia coli MRE 600 (EC 6.1.1.5) and various amino acids have been determined calorimetrically in 50 mM potassium phosphate buffer, at pH 7.5, in the presence of 1 mM dithioerythritol. The delta Ha values for binding of L-isoleucine, L-leucine, L-valine, L-norvaline and L-2-amino-3S, 4-dimethyl pentanoic acid agree within the limits of experimental error in magnitude (3.7 +/- 0.9 kcal mol-1 or 15.5 +/- 3.8 kJ mol-1 at 25 degrees C) and variation with temperature (delta cp = -430 +/- 20 cal mol-1 K-1 or 1799 +/- 84 J mol-1 K-1). In view of the large differences in the equilibrium constants for the corresponding binding equilibria, the identical association enthalpies suggest that the enthalpic contribution to the Gibbs free energy of binding, delta Ga, cannot be responsible for the specificity of the interaction of the enzyme with the amino acids. It has rather to be inferred that the entropic term, delta Sa, is decisive in discriminating the correct amino acid. Analogous calorimetric binding studies on the reaction between L-isoleucinol and the enzyme suggest that the absence of the carboxyl group renders the association enthalpy more positive (by 4-5 kcal mol-1 or 16.7-20.9 kJ mol-1) with respect to that of the amino acids. The variation with temperature of the delta Ha values, however, practically parallels that of the amino acids.

The binding of tRNAIIe to isoleucyl-tRNA synthetase in the presence of isoleucine or ATP was investigated using the equilibrium partition method. Isoleucine decreased the affinity of tRNAIIe for the enzyme by a factor of about 5. For the free standard energy of interaction a value of about 1 kcal/mol (4.2 kJ/mol) was calculated. ATP exhibits qualitatively the same effect as isoleucine. A binding of two molecules isoleucine per molecule of enzyme could not be demonstrated even in the presence of ATP and pyrophosphatase.