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

Hertz, Harry S.; Zachau, Hans G.

doi:10.1111/j.1432-1033.1973.tb02977.x

Cited by 22 publications

(3 citation statements)

References 46 publications

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“…Serine was found to occupy more than one binding site also at low concentrations which agrees with the earlier finding of non-linear Lineweaver-Burk plots (18). On the other hand, it was shown in the present study that only one serine molecule becomes activated even in the presence of pyrophosphatase (Fig.…”

Section: C0supporting

confidence: 93%

Serine activation is the rate limiting step of tRNA^Seraminoacylation by yeast seryl tRNA synthetase

1980

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Using the quenched flow technique the mechanism of seryl tRNA synthetase action has been investigated with respect to the presteady state kinetics of individual steps. Under conditions where the strong binding sites of the enzyme are nearly saturated and the steady state turnover number is about 1 s-1, rate constants of four different processes have been determined: steps connected with substrate associations are relatively slow (12 s-1 for the entire process); activation of serine is the rate determining step (about 1.2 s-1 in presence of tRNASer); whereas the transfer of serine onto tRNASer (35 s-1) and the dissociation of seryl tRNASer (70 s-1) are fast. Similar kinetic parameter seem to hold also for the steady state reactions. This conclusion is based on a detailed study of the substrate, product, and Mg2+ concentration dependence of the transfer reaction. The results also indicate that a second serine binding site is operative. Since the transfer of serine from a preformed adenylate complex onto tRNASer is fast, seryl adenylate seems to be a kinetically competent intermediate of the aminoacylation reaction although, of course, alternative mechanisms cannot be excluded.

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Section: C0supporting

confidence: 93%

Serine activation is the rate limiting step of tRNA^Seraminoacylation by yeast seryl tRNA synthetase

1980

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“…Seryl tRNA synthetase seems to share reaction kinetic features with a number of other aminoacyl tRNA synthetases. A second order dependence of the reaction rate on the tRNA concentration as it was previously demonstrated with the serine enzyme (22,23) and is investigated in more detail in the present study, has also been found with the leucine and tryptophan enzymes from E. coli and beef pancreas (23), respectively. Furthermore, one form of glycyl tRNA synthetase from E. coli shows a Hill coefficient of 2 with tRNA as the varied substrate (24).…”

Section: Discussionsupporting

confidence: 75%

Yeast seryl tRNA synthetase: two sets of substrate sites involved in aminoacylation

Pachmann

Zachau

1978

Nucl Acids Res

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Seryl tRNA synthetase from Saccharomyces Carlsbergensis C836 contains two sets of sites for tRNASer, L-serine, and Mg2+-ATP, both of which are involved in aminoacylation. This is based on the following experimental results: (a) at low serine concentrations, second order kinetics in tRNASer are observed; (b) biphasic kinetics result when the amino acid is the varied substrate indicating anticooperative binding of two serine molecules to the synthetase; (c) when two molecules of serine are bound the rate of aminoacylation increases strongly and becomes first order in tRNASer; (d) the involvement of more than one site for Mg2+ and ATP is deduced from systematic variations of the concentrations of Mg2+ and ATP. Implications of the anticooperative binding of the substrates for possible reaction mechanisms are discussed. The results indicate that under normal conditions, the activity of seryl tRNA synthetase is regulated mainly by tRNASer while at high serine concentrations regulation by the amino acid itself prevails.

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“…Even a t pH 13.0 (0.1 N NaOH), where &-adenosine decomposes rapidly, it was possible to perform fluorescence lifetime measurements within minutes of sample preparation, and the lifetime was found to be 20 ns. If the observations a t lower pH values are not considered sufficient to rule out the participation of the protonatcd form, the observations a t high pH could be consistent either with emission by the unprotonated form 1 or with a protonated form having an excited state pKa no less than 14 or 15, since no appreciable decrease of the yield was seen a t pH 13.0.…”

Section: P H Dependence Of Fluorescence Lifetimesmentioning

confidence: 99%

Species Responsible for the Fluorescence of 1: N6-Ethenoadenosine

et al. 1974

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1. Fluorescence lifetimes and emission spectra of 1 : N6-ethenoadenosine 5'-monophosphate (&) in aqueous solution over the pH range 1.5 to 12.0 indicate the presence of only one emitting fluorophore.2. The loss a t low pH of fluorescence emission a t 415 nm from the neutral 1 : N6-ethenoadenine fluorophore is due to the conversion of the fluorescent unprotonated form to the non-fluorescent protonated form by protonation a t N-9. This conclusion is based on the pH dependence of the fluorescence lifetimes and quantum efficiences of CAMP over the range 1.5 to 12.0.3. The observation of a fluorescence quantum efficiency of 86O/, that of & in aqueous solution (pH 6.8) for 1 : N6-etheno-9-propyladenine (E-PrAde) in dry dioxane where it cannot acquire a proton in the excited state is direct evidence that the unprotonated form of the Eadenine fluorophore is responsible for the fluorescence emission. the interpretation of the fluorescence data obtained necessitate an understanding of the excited state of the fluorophore, since quantum efficiencies, lifetimes and spectra reflect environmental conditions surrounding the fluorophore. It is our purpose here to define the excited state of 1 : N6-ethenoadenosine, especially since Penzer [14] has proposed that in aqueous solutions fluorescence is emitted from the 9-protonated form of I a .I n the initial description of the fluorescence properties of 1 : N6-ethenoadenosine [l], evidence was presented that the neutral form of &Ado is responsible for the fluorescence. Emission spectra taken a t intervals between pH 7.0 and 2.2 showed that the emission maximum a t 415 nm remained constant over this pH range while the fluorescence intensity decreased progressively beginning about pH 5.5 and continuing until, at pH 2.2, the intensity was less than loo/, of the intensity a t pH 7.0. The fluorescence excitation spectrum indicated that the long-wavelength band centered at 300 nm was responsible for fluorescence. The disappearance of the long-wavelength band in the absorption spectrum with the loss of fluorescence intensity a t 415 nm as I : N6-ethenoadenosine (pKa = 3.9) becomes protonated, suggested that only one structure, the neutral form, is the fluorophore [l].Nevertheless, Penzer has proposed that although the pKa of the ground state protonation is known to be 3.9 [l], the excited state pKa is five or more units more basic and that upon excitation, a proton is donated by water: &Ado* + H,O z &Ado*-H+ + OH-.(1) Eur. J. Biochem. 45 (1974)

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

Cited by 22 publications

References 46 publications

Serine activation is the rate limiting step of tRNA^Seraminoacylation by yeast seryl tRNA synthetase

Serine activation is the rate limiting step of tRNA^Seraminoacylation by yeast seryl tRNA synthetase

Yeast seryl tRNA synthetase: two sets of substrate sites involved in aminoacylation

Species Responsible for the Fluorescence of 1: N6-Ethenoadenosine

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

Cited by 22 publications

References 46 publications

Serine activation is the rate limiting step of tRNASeraminoacylation by yeast seryl tRNA synthetase

Serine activation is the rate limiting step of tRNASeraminoacylation by yeast seryl tRNA synthetase

Yeast seryl tRNA synthetase: two sets of substrate sites involved in aminoacylation

Species Responsible for the Fluorescence of 1: N6-Ethenoadenosine

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Serine activation is the rate limiting step of tRNA^Seraminoacylation by yeast seryl tRNA synthetase

Serine activation is the rate limiting step of tRNA^Seraminoacylation by yeast seryl tRNA synthetase