On the non‐liner Eadie plots of the tRNA kinetics and non‐liner Dixon plots of the PP_i inhibition kinetics of the aminoacyl‐tRNA synthetases

Abstract

A model of the aminoacyl-tRNA synthetase reaction was analyzed by deriving a rate equation, and by calculating the aminoacylation rates at various values of the rate and equilibrium constants. The model specially contained the possibilities that (1) the activation of the amino acid occurs either with bound or non-bound tRNA, and that (2) the transfer of the aminoacyl moiety from the aminoacyl adenylate to tRNA occurs either with bound or non-bound PPi. The analysis showed that the Eadie plots (tRNA as the variable substrate) are straight lines only if the rates of the activation reactions with bound and non-bound tRNA are equal. Otherwise the Eadie plots can be either curved upwards or downwards. The Dixon plots of the PPi inhibition are straight lines only if PPi must be dissociated from the enzyme before the transfer reaction. The conditions under which the KfPP values are much lower than the dissociation constants for PPi are met if the transfer reaction is relatively slow and the reverse reaction of the activation (pyrophosphorolysis) is fast, and if the tRNA concentration is low.The aminoacylation of tRNA by the aminoacyl-tRNA synthetases involves two main steps, the activation of the amino acid by ATP and the transfer of the activated amino acid to tRNA [l -31. In the activation an aminoacyl adenylate is formed and PPi is released, but two separate possibilities still exist: the tRNA could be bound to the enzyme before the activation or after it. Also in the transfer step two separate possibilities exist: the transfer reaction might take place in the presence of an enzyme-bound PPi molecule or in its absence.It is possible, or even probable, that the rates of the activation are different in the presence and in the absence of the bound tRNA molecule. Also the rates of the transfer reaction may be different in the presence and in the absence of PPi. A kind of conformational activation has been reported upon binding of tRNA [4]. Our previous measurements of the aminoacylation of tRNA by the Leu-tRNA synthetase from Neurospora crassa showed Eadie plots curved upwards for the tRNA kinetics [5]. The plots could be satisfactorily fitted by a kinetic model where the rates of the activation reactions were different from each other. Another abnormality was found with the isoleucyl-tRNA synthetase from Escherichiu coli, where the Eadie plots of the tRNA kinetics were curved downwards. Similar non-linear Eadie plots have been found also for the tRNA kinetics of the Ile-tRNA synthetase from yeast [6].The kinetics of the PPi inhibition of the aminoacylation of tRNA by the Leu-tRNA synthetase from N . crassa gave always Dixon plots ( l / v vs [PPi]) curved upwards, and the K:pp values were very low [7]. A kinetic model derived to describe the PPi inhibition of this enzyme gave low K, values but could not explain the curved Dixon plots.In the present work a steady-state kinetic model for the aminoacylation of tRNA by the aminoacyl-tRNA synthetases METHODSThe Ile-tRNA synthetase and tRNATle preparations as well as the met...