Amino acid dependent ATP—<sup>32</sup>PP<sub>i</sub> exchange measurement a filter paper disk method

Simlot, M. M.; Pfaender, Peter

doi:10.1016/0014-5793(73)80284-4

Cited by 21 publications

(11 citation statements)

References 8 publications

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“…69/K, 2.2 cm diameter, J. C. Binzer, Hatzfeld/Eder, Germany). Washing and liquid scintillation counting of the filters was performed as described (14). All velocities are given in cpm.…”

mentioning

confidence: 99%

Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species.

Haar¹,

Gaertner²

1975

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACTtRNAPhe species from baker's yeast modified at the 3'-terminus in many cases are phenylalanylatable substrates. Out of several tRNAPhe species possessing a modified 3'-end that cannot be phenylalanylated, only two, tRNAPheC-C-2'dA and tRNAPheC-C-formycinoxi-reds are strong competitive inhibitors for tRNAPheC-C-A during phenylalanylation. In the ATP/PPi exchange, both these inhibitors reduce Vman, to about 25%o; but whereas tRNAPheCC_2'dA has no influence on Km ATP and Km Phe during ATP/PPj exchange, tRNAPheCC-formycinox i-red reduces Km AT! from 1430 IAM, found in the absence of tRNAPhe, to 230 jiM, and Km Phe, from 38 to 14 uM. The values found in the presence of tRNAPheC-C-CformycinOxi-re during ATP/PPi exchange, are identical with those determined in the phenylalanylation of tRNAPheC_C-A.All other tRNAPhe species carrying a modified 3'-end that cannot be phenylalanylated exhibit a mixed competitivenoncompetitive inhibition in the phenylalanylation reaction. In the ATP/PPi exchange, they do not influence Km ATP and K,, Phe and only weakly, if at all, Vmax.The results show that the 3'-adenosine of tRNAPhe cannot solely be a passive acceptor for phenylalanine, but must in addition play an active role during enzyme-substrate interaction. The data can be consistently explained by the hypothesis that the 3'-adenosine of tRNAPhe triggers a conformational change of the enzyme.In the living cell part of the genetic information stored in the DNA is translated into proteins. This is achieved by first transcribing the information into mRNAs and then translating the latter into peptide sequences on the ribosome. Prior to translation on the ribosome, the amino acids are activated by attachment to the 3'-terminal ribose of their particular tRNAs.The overall aminoacylation reaction is achieved according to tRNAaa + ATP + aa + E 2 aa-tRNAaa + AMNP + PPj + E, where E is the enzyme and aa is the amino acid. This reaction can be followed experimentally by the incorporation of radioactively labeled amino acid into tRNA.

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“…69/K, 2.2 cm diameter, J. C. Binzer, Hatzfeld/Eder, Germany). Washing and liquid scintillation counting of the filters was performed as described (14). All velocities are given in cpm.…”

mentioning

confidence: 99%

Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species.

Haar¹,

Gaertner²

1975

Proc. Natl. Acad. Sci. U.S.A.

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“…To determine the active concentration of functional enzyme, we used an active site titration procedure as described by Francklyn et al [55] with some modifications. Aliquots were transferred at varying time points onto activated charcoal filters (69 ⁄ K, diameter 25 mm; Munktell and Filtrak, Ba¨renstein, Germany) [56] and the filters were washed twice with 1.5% perchloric acid containing 40 mm sodium pyrophosphate and once with water. After brief drying, they were counted in a scintillation counter.…”

Section: Methodsmentioning

confidence: 99%

The influence of identity elements on the aminoacylation of tRNA^Arg by plant and Escherichia coli arginyl‐tRNA synthetases

2012

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Identity elements determine the accurate recognition between tRNAs and aminoacyl-tRNA synthetases. The arginine system from yeast and Escherichia coli has been studied extensively in the past. However, information about the enzymes from higher eukaryotes is limited and plant aminoacyltRNA synthetases have been largely ignored in this respect. We have designed in vitro tRNA transcripts, based on the soybean tRNA Arg primary structure, aiming to investigate its specific aminoacylation by two recombinant plant arginyl-tRNA synthetases and to compare this with the enzyme from E. coli. Identity elements at positions 20 and 35 in plants parallel those previously established for bacteria. Cryptic identity elements in the plant system that are not revealed within a tRNA Arg consensus sequence compiled from isodecoders corresponding to nine distinct cytoplasmic, mitochondrial and plastid isoaccepting sequences are located in the acceptor stem. Additionally, it has been shown that U20a and A38 are essential for a fully efficient cognate E. coli arginylation, whereas, for the plant arginyl-tRNA synthetases, these bases can be replaced by G20a and C38 with full retention of activity. G10, a constituent of the 10 : 25 : 45 tertiary interaction, is essential for both plant and E. coli activity. Amino acid recognition in terms of discriminating between arginine and canavanine by the arginyl-tRNA synthetase from both kingdoms may be manipulated by changes at different sites within the tRNA structure.

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“…Separation of [35S]ATPaS (A) from [35S]AMPS was achieved on poly(ethy1eneimine)-cellulose plates according to [13]. ATP/PPi exchange reaction was performed as described [11,14]. Reaction was followed at 31 "C in 100-p1 assay mixtures containing 150 mM Tris .…”

Section: Methodsmentioning

confidence: 99%

On the Stereochemistry of Activation of Phenylalanine by Phenylalanyl‐tRNA Synthetase from Baker's Yeast

Haar¹,

Cramer²,

Eckstein³

et al. 1977

European Journal of Biochemistry

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Because of its chiralic a-phosphorus atom adenosine 5'-0-( 1 -thiotriphosphate) (ATPaS) exists in two diastereomeric forms, arbitrarily named (A) and (B).For phenylalanyl-tRNA synthetase ATPaS (A) is a substrate whereas ATPaS (B) is neither a substrate nor an inhibitor. During the ATPaS (A)/PPi exchange reaction with phenylalanyl-tRNA synthetase the configuration at the a-phosphorus atom is retained. The mechanistic implications of these findings are discussed.Preliminary investigations with several other aminoacyl-tRNA synthetases show that the stereochemical requirement with respect to the a-phosphorus of ATP is not identical for all aminoacyltRNA synthetases.Aminoacyl-tRNA synthetases catalyze the transfer of an amino acid onto a transfer ribonucleic acid. They use ATP to activate their respective amino acid by formation of a mixed anhydride between these two substrates (reaction a). The amino acid is subsequently transferred to the third substrate, the tRNA (reaction b) [I -31. aa + ATP e aa-AMP + PPi Various ATP analogues have been used to study the specificity as well as to establish the mechanism of this reaction [4,5]. Important insight into the mechanism of activation (reaction a) can be obtained by studying the pyrophosphate exchange reaction. In addition, analogues of ATP such as the diastereomers of adenosine 5'-O-(l-thiotriphosphate) [6] can yield information on the stereospecificity as well as the stereochemistry of this reaction. In this report we would like to describe such studies with aminoacyltRNA synthetase, in particular the phenylalanyltRNA synthetase from baker's yeast.Abbreviations. ATPctS (A) and ATPaS (B) = adenosine 5'-0-(I-thiotriphosphate) (diastereomers arbitrarily named); tRNAPhr-C-C-FoXi-,.d = tRNAPhe with terminal oxidized and reduced formycin instead of adenosine; EPhe = phenylalanyl-tRNA synthetase.

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Amino acid dependent ATP—³²PP_i exchange measurement a filter paper disk method

Cited by 21 publications

References 8 publications

Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species.

Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species.

The influence of identity elements on the aminoacylation of tRNA^Arg by plant and Escherichia coli arginyl‐tRNA synthetases

On the Stereochemistry of Activation of Phenylalanine by Phenylalanyl‐tRNA Synthetase from Baker's Yeast

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Amino acid dependent ATP—32PPi exchange measurement a filter paper disk method

Cited by 21 publications

References 8 publications

Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species.

Phenylalanyl-tRNA synthetase from baker's yeast: role of 3'-terminal adenosine of tRNA-Phe in enzyme-substrate interaction studied with 3'-modified tRNA-Phe species.

The influence of identity elements on the aminoacylation of tRNAArg by plant and Escherichia coli arginyl‐tRNA synthetases

On the Stereochemistry of Activation of Phenylalanine by Phenylalanyl‐tRNA Synthetase from Baker's Yeast

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Amino acid dependent ATP—³²PP_i exchange measurement a filter paper disk method

The influence of identity elements on the aminoacylation of tRNA^Arg by plant and Escherichia coli arginyl‐tRNA synthetases