Kinetic Properties of Phenylalanyl‐tRNA and Seryl‐tRNA Synthetases for Normal Substrates and Fluorescent Analogs

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Abstract
The kinetics of phenylalanyl‐tRNA and seryl‐tRNA formation were investigated with tRNAs and aminoacyl‐tRNA synthetases from yeast.Phenylalanyl‐tRNA synthetase yielded linear Lineweaver‐Burk plots with tRNAphe, phenylalanine, and 1,N6‐ethenoadenosine triphosphate (ɛATP) as variable substrates. According to equilibrium dialysis in the absence or presence of phenylalaninyl adenosine 5′‐phosphate, phenylalanyl‐tRNA synthetase possesses one binding site for phenylalanine. For ATP as variable substrate, the deviation from linearity in the Lineweaver‐Burk plot, observed by other investigators, was confirmed. The slope of the curve indicates the presence of more than two ATP binding sites.Seryl‐tRNA synthetase yielded a linear Lineweaver‐Burk plot only with ɛATP as variable substrate. The Lineweaver‐Burk plots for serine and tRNASerwere non‐linear; the interpretation we favor involves positive cooperativity between amino acid binding sites and between tRNA binding sites. Hill plots of the kinetic data showed that the enzyme possesses at least two binding sites for each of these substrates. The kinetic data for ATP could be interpreted as showing more than two binding sites with negative and positive cooperativity in binding of successive ATP molecules.The aminoalkyl adenylates, phenylalaninyl adenosine 5′‐phosphate and serinyl adenosine 5′‐phosphate, competitively inhibited the aminoacylation reaction with respect to amino acid.ɛATP functions in place of ATP in phenylalanyl‐tRNA and seryl‐tRNA formation although with rather different kinetic properties. Modified tRNApheand tRNASer, in which the 3′‐terminal adenosine was replaced by ethenoadenosine, were prepared by a C‐C‐A transferase‐catalyzed reaction of ɛATP. These modified tRNAs show kinetic properties very similar to those of the unmodified tRNAs and can therefore be used, in place of the unmodified tRNAs, as fluorescent probes in synthetase‐tRNA interaction studies.The kinetic behavior of phenylalanyl‐tRNA synthetase appears to be much simpler than that of seryl‐tRNA synthetase, despite the fact that the former enzyme is twice as big and contains twice as many subunits as the latter one. The comparative simplicity of the one enzyme relative to the other correlates with previous results on interactions with substrates, which were obtained by fluorescence measurements and nuclease protection studies.