Abstract
Kirromycin induces a GTPase activity dependent on elongation factor Tu (EF-Tu) in the absence of the normally required aminoacyl-tRNA (aa-tRNA) and ribosomes [Wolf et al. (1974) Proc. Natl Acad. Sci. USA, 71, 4910–4914]; these two components however stimulate the turnover reaction, which is strongly affected by monovalent cations [Sander et al. (1979) FEBS Lett. 98, 111–114]. In this work we have studied the influence of the temperature on the GTP hydrolysis catalyzed by EF-Tu · kirromycin in the presence or absence of aa-tRNA and/or ribosomes and at two concentrations of monovalent cations (30 and 200 mM NH4Cl). This was possible, since we have found that under all these conditions, the GTP breakdown is the rate-limiting step, even though the rate of the turnover reaction shows great variations. The determination of the energetic parameters of the GTP hydrolysis was made by Arrhenius plots that gave straight lines over a broad range of temperature. The presence of aa-tRNA and/or ribosomes and different NH4Cl concentrations do not substantially affect the energy of activation, the values all being in the range 67–78 KJ · Mol−1 The entropy of activation is increased by ribosomes only at low salt conditions (30 mM NH4Cl). Higher concentrations of salts (200 mM NH4Cl or 2 M LiCl) increase the entropy of activation indicating that monovalent cations can substitute partially for ribosomes. The similar values of the energy of activation, as well as of the Km, values, which in the different conditions were all in the range of 0.2–0.4 μM, support the conclusion that in the presence of kirromycin the catalytic center of the GTPase activity in the different systems is entirely located on the EF-Tu molecule. The Arrhenius plots derived from the GTPase activity of EF-Tu in the absence of kirromycin, and thus dependent upon the presence of aa-tRNA and mRNA · ribosomes, are much more complicated and at present are difficult to interpret.
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