Interactions of seryl‐tRNA synthetase with serine and phenylalanine specific tRNA

Rigler, Rudolf; Cronvall, E.; Hirsch, Rolf D.; Pachmann, Ulrich; Zachau, Hans G.

doi:10.1016/0014-5793(70)80558-0

Cited by 54 publications

(42 citation statements)

References 9 publications

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“…The experimental parameters were found to differ considerably from the standard test conditions [4,33f. that V values obtained a t the enzyme concentration required for the initial rate studies can be approximately doubled when the enzyme concentration range is raised above 10 nM [5].…”

Section: Discussionmentioning

confidence: 86%

“…From the n values obtained from the Hill plots, cooperativity is also indicated, with a minimum of two binding sites for each of the two substrates. In fluorescence studies [5], cooperativity has been previously indicated for the binding of tRNAser to seryl-tRNA synthetase. I n studies of protection against nuclease digestion [S] and in fluorescence measurements [5,7,39] two sites for tRNAser were obtained.…”

Section: Discussionmentioning

confidence: 98%

“…In fluorescence studies [5], cooperativity has been previously indicated for the binding of tRNAser to seryl-tRNA synthetase. I n studies of protection against nuclease digestion [S] and in fluorescence measurements [5,7,39] two sites for tRNAser were obtained. Unfortunately attempts to determine serine binding sites by equilibrium dialysis were unsuccessful, due to the low binding constant (which correlates with the relatively high "Rm").…”

Section: Discussionmentioning

confidence: 98%

“…Phenylalanyl-tRNA synthetase and seryl-tRNA synthetase from yeast were prepared in cooperation with R. Hirsch as described previously [3 -5,151 (and R. Hirsch, unpublished work) and had the previously described properties [4,5] ; some samples were generously provided by R. Hirsch. One A,,, unit phenylalanyl-tRNA synthetase and seryltRNA synthetase were taken to be 1 mg and 0.82 mg, respectively.…”

Section: Methodsmentioning

confidence: 99%

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

Hertz¹,

Zachau²

1973

European Journal of Biochemistry

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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 ,Ne-ethenoadenosine triphosphate ( EATP) 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-tBNA spthetase yielded a linear Lineweaver-Burk plot only with EATP as variable substrate. The Lineweaver-Burk plots for serine and tRNAS-9' were 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 a t 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 6'-phosphate and serinyl adenosine 5'-phosphate, competitively inhibited the aminoacylation reaction with respect to amino acid.EATP functions in place of ATP in phenylalanyl-tRNA and seryl-tRNA formation although with rather Werent kinetic properties. Modified tRNAPhe and tRNASer, in which the 3'-terminal adenosine was replaced by ethenoadenosine, were prepared by a C-C-A transferase-catalyzed reaction of EATP. These modified tRNAs show kinetic properties very similar to those of the unmodified tRNAs and can therefore be used, in place of the unmodsed 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 synthetaae, 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.

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Section: Discussionmentioning

confidence: 86%

Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

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

Hertz¹,

Zachau²

1973

European Journal of Biochemistry

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“…3 G(ko/k) In the experiments where aminoacyl-tRNA-synthetases were titrated with tRNA the fluorescence intensity of the enzyme was measured together with the intensity of transmitted light ( fig. 2a, c in the following paper [6]). In this way kd is directly measured and changes in the extinction coefficients caused by tRNA-synthetase interaction can be accounted for.…”

Section: Theory and Resultsmentioning

confidence: 91%