Binding of tyrosine, adenosine triphosphate and analogues to crystalline tyrosyl transfer RNA synthetase

Monteilhet, Claude; Blow, D. M.

doi:10.1016/0022-2836(78)90418-7

Cited by 61 publications

(25 citation statements)

References 24 publications

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“…X-ray analysis of the enzyme from B. stearothermophilus revealed that the tyrosine-binding site is a pocket surrounded on all sides by protein but does not have a 'superspecificity' feature which would allow tyrosine recognition in a single step [48]. An unexpected feature of the groove is that 'it is far from hydrophobic' [48], the upper surface of the groove having several amide and hydroxyl groups.…”

Section: Discussionmentioning

confidence: 99%

“…An unexpected feature of the groove is that 'it is far from hydrophobic' [48], the upper surface of the groove having several amide and hydroxyl groups. These results are consistent with our AACl, and AdGI, values.…”

Section: Discussionmentioning

confidence: 99%

“…Four-step recognition process of amino acids by tyrosyl-tRNA synthetase the cavity. The binding pocket can accept planar groups with different chemical features such as adenine [48,491. This makes it easier to understand that amino acids with longer side chains such as arginine and tryptophan are transferred in misacylation reactions to tRNATy'.…”

Section: Discussionmentioning

confidence: 99%

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Tyrosyl‐tRNA synthetase from baker's yeast

Freist

Sternbach

1988

European Journal of Biochemistry

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The order of substrate addition to tyrosyl-tRNA synthetase from baker's yeast was investigated by bisubstrate kinetics, product inhibition and inhibition by dead-end inhibitors. The kinetic patterns are consistent with a random bi-uni uni-bi ping-pong mechanism.Substrate specificity with regard to ATP analogs shows that the hydroxyl groups of the ribose moiety and the amino group in position 6 of the base are essential for recognition of ATP as substrate.Specificity with regard to amino acids is characterized by discrimination factors D which are calculated from k,,, and K , values obtained in aminoacylation of tRNATY'-C-C-A. The lowest values are observed for Cys, Phe, Trp (D = 28000-40000), showing that, at the same amino acid concentrations, tyrosine is 28000-40000 times more often attached to tRNATY'-C-C-A than the noncognate amino acids. With Gly, Ala and Ser no misacylation could be detected (D > 500000); D values of the other amino acids are in the range of 100000-500000.Lower specificity is observed in aminoacylation of the modified substrate tRNATYr-C-C-A(3'NH2) ( D , = 500 -55000). From kinetic constants and AMP-formation stoichiometry observed in aminoacylation of this tRNA species, as well as in acylating tRNATYr-C-C-A hydrolytic proof-reading Factors could be calculated for a pretransfer ( H I ) and a post-transfer (n,) proof-reading step. The observed values of n , = 12-280 show that pretransfer proof-reading is the main correction step whereas post-transfer proof-reading is marginal for most amino acids (n, = 1-2).Initial discrimination factors caused by differences in Gibbs free energies of binding between tyrosine and noncognate amino acids are calculated from discrimination and proof-reading factors. Assuming a two-step binding process, two factors ( I , and I , ) are determined which can be related to hydrophobic interaction forces. The tyrosine side chain is bound by hydrophobic forces and hydrogen bonds formed by its hydroxyl group. A hypothetical model of the amino acid binding site is discussed and compared with results of X-ray analysis of the enzyme from Bacillus .strurothcrmophilus.Tyrosyl-tRNA synthetases represent a group of aminoacyl-tRNA synthetases which has been investigated very intensively and detailed. Complete primary structures are known for the enzymes from Escherichia Cali and Bacillus stenrothermophilus [l, 21, exhibiting an amino acid sequence homology of 56% [2]. Tyrosyl-tRNA synthetase from B. strarothermophilus was crystallized and analyzed by X-ray diffraction as free enzyme [3] and complexed with tyrosyladenylate [4], which is the enzyme-bound product of the first reaction step in the aminoacylation reaction : E + ATP + Tyr + E . Tyr-AMP + PP, E . Tyr-AMP + tRNA + E + Tyr-tRNA + AMP.From X-ray analyses several hydrogen bonds between amino acid side chains of the enzyme and tyrosyladenylate were postulated [5, 61 and a considerable number of mutant enCorrespondence to W. Freist, Abteihng Chemie,

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Tyrosyl‐tRNA synthetase from baker's yeast

Freist

Sternbach

1988

European Journal of Biochemistry

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“…For tyrosyl-tRNA synthetase from B. stParothernzol,hilus it was shown by electron density difference maps that, in complexes obtained with crystalline enzyme and AMP or tyrosine, surprisingly these two compounds bind at the same site, and tyrosinyl-adenylate is also bound with the tyrosyl side chain to that same site [47]. Obviously two hydrophobic pockets must exist for binding of the aminoacyladenylate, one for the sidc chain of the amino acid and one for the adenine moiety.…”

Section: Speculations and Experiments The Substrate Binding Pocket Rmentioning

confidence: 99%

Isoleucyl‐tRNA Synthetase from Baker's Yeast

Freist

Cramer

1983

European Journal of Biochemistry

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The aminoacylation of three modified tRNAIle species with isoleucine and with valine by isoleucyl‐tRNA synthetase has been investigated by initial rate kinetics. For aminoacylation of tRNAIle‐C‐C‐3′dA with isoleucine, a bi‐bi uni‐uni ping‐pong mechanism has been found by bisubstrate kinetics and inhibition by products and by 3′dATP; for aminoacylation with valine a bi‐uni uni‐bi ping‐pong mechanism. For isoleucylation of tRNAIle‐C‐C‐A(3′NH2) bisubstrate kinetics, inhibition by products and by isoleucinol show a random uni‐bi uni‐uni‐uni ping‐pong mechanism; for valylation of this tRNA a bi‐bi uni‐uni ping‐pong mechanism is observed by bisubstrate kinetics and product inhibition. tRNAIle‐C‐C‐2′dA was aminoacylated under modified conditions with isoleucine in a bi‐bi uni‐uni ping‐pong mechanism with a rapid equilibrium segment as observed by bisubstrate kinetics, inhibition by AMP, by P[NH]P as product analog and by isoleucinol. Aminoacylation with valine is achieved in a rapid equilibrium sequential random AB, ordered C mechanism indicated by bisubstrate kinetics and inhibition by 3′dATP and valinol. All six reactions exhibit orders of substrate addition and product release which are different from those observed in aminoacylation of the natural tRNAIle‐C‐C‐A. The Km values of the three substrates and the kcat values of the six reactions are given. For aminoacylation at the terminal 2′OH group of the tRNA differences of 13.38 and 13.17 kJ in binding energies between valine and isoleucine have been calculated which result in discrimination factors of 181 and 167. For aminoacylation at the terminal 3′‐OH group a differnce of only 4.43 kJ and a low discrimination factor of only 6 is observed. Thus maximal discrimination between the cognate and the noncognate amino acid is only achieved in aminoacylation at the 2′‐OH group and conclusions drawn from experiments with modified tRNAs concerning 2′,3′‐specificity have led to correct results in spite of different catalytic cycles in aminoacylation of the natural and the modified tRNA. The stability of Ile‐tRNAIle‐C‐C‐2′dA and Val‐tRNAIle‐C‐C‐2′dA, the lesser stability of Val‐tRNAVal‐C‐C‐2′dA and the instability of Thr‐tRNAVal‐C‐C‐2′dA are consistent with postulations for a ‘pre‐transfer′ proofreading step for isoleucyl‐tRNA synthetase and a ‘post‐transfer’ hydrolytic editing step for valyl‐tRNA synthetase at the terminal 3′OH group of the tRNA.

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“…Structure amplitudes had also been obtained for crystals which had been treated with the inhibitor tyrosinyl adenylate (Monteilhet & Blow, 1978), and for crystals in which the intermediate on the catalytic pathway, tyrosyl adenylate, was formed (Rubin & Blow, 1981). Tyrosinyl adenylate gave an electrondensity difference map which was clearly interpretable, with the isomorphous replacement phase angles, while the difference map for tyrosyl adenylate was much more noisy.…”

Section: Applicationmentioning

confidence: 99%

A density-modification method for the improvement of poorly resolved protein electron-density maps

Bhat¹,

Blow²

1982

Acta Cryst Sect A

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An efficient computer procedure has been developed for the extraction of regions of contiguous, wellconnected high density from a three-dimensional electron-density map. This procedure may be used to generate an extended model volume, from a smaller volume based on a starting atomic model. The starting model may, for example, only include main-chain atoms for a protein, or may omit uninterpretable segments of chain. The procedure may also be used to extract regions of contiguous density where no model exists. The extended model volume is used to produce a properly scaled model electron density. Calculated structure factors are obtained from the scaled model electron density by fast Fourier transform, and combined, with appropriate weights, with the existing phase information to give improved phase angles. Calculation of a new electron-density map with the new phase angles initiates the next step of a cyclic procedure which converges rapidly. The procedure has been applied to the structure determination of tyrosyl-tRNA synthetase. It has led to identification of most of the available amino-acid sequence in the electron density, and a revised tracing of the main polypeptide chain. Evidence for improvement in phase angles is obtained from electron-density difference maps for substrate and inhibitor binding, in which a reduction in background density is observed.

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Binding of tyrosine, adenosine triphosphate and analogues to crystalline tyrosyl transfer RNA synthetase

Cited by 61 publications

References 24 publications

Tyrosyl‐tRNA synthetase from baker's yeast

Tyrosyl‐tRNA synthetase from baker's yeast

Isoleucyl‐tRNA Synthetase from Baker's Yeast

A density-modification method for the improvement of poorly resolved protein electron-density maps

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