Ionic Strength Effects in the Aminoacylation of Valine Transfer Ribonucleic Acid

Loftfield, Robert B.; Eigner, Elizabeth Ann

doi:10.1016/s0021-9258(18)99435-4

Cited by 54 publications

(14 citation statements)

References 8 publications

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“…of the tRNA-independent and -dependent activities, respectively, is observed. It should be noted that 0.3 M KC1 completely abolishes the aminoacylation of tRNA catalyzed by valyl-tRNA synthetase (Jakubowski & PawejJtiewicz, 1977), most likely by removing tRNA from the enzyme as has been shown for E. coli valyl-tRNA synthetase (Loftfield & Eigner, 1967). Thus, at high salt concentrations the tRNA-dependent activity should become tRNA independent, which is actually seen in Figure 6.…”

Section: Resultsmentioning

confidence: 73%

“…First, the ATP pyrophosphohydrolase activity in the absence of tRNA is not affected by RNase, whereas the reaction in the presence of tRNA is suppressed by RNase to the level observed in the absence of tRNA. Second, this activity persists under the conditions which lead to complete dissociation of tRNA from the enzyme, e.g., at high pH values or in the presence of high salt concentrations (Loftfield & Eigner, 1967; Lam & Schimmel, 1975). Third, the apparent ATP pyrophosphohydrolase activity is not inhibited by periodate oxidized tRNA, which still is able to bind to the enzyme (Table IV).…”

Section: Discussionmentioning

confidence: 99%

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Valyl-tRNA synthetase from yellow lupin seeds: hydrolysis of the enzyme-bound noncognate aminoacyl adenylate as a possible mechanism of increasing specificity of the aminoacyl-tRNA synthetase

Jakubowski¹

1980

Biochemistry

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This activity is due to hydrolysis of the enzyme-bound noncognate aminoacyl adenylates. The enzyme-bound valyl adenylate is apparently protected by the enzyme and slowly decomposes with a rate constant of 0.018 min-1, which is 720 times slower than the rate constant of hydrolysis of valyl-tRNA synthetase-bound cysteinyl adenylate (k = 13 min-1). The KM value for ATP in the ATP pyrophosphohydrolase reaction is 9 µ and does not depend on the nature of the amino acid. This value is one order of magnitude lower than the KM value for ATP in the reaction of tRNA aminoacylation with valine (ÁTM = 100 µ ). The cysteine-dependent ATP pyrophosphohydrolase activity of valyl-tRNA synthetase in the absence of tRNA exhibits pH optimum between 9.5 and 10.5 in glycine-NaOH buffer, is moderately sensitive to KC1 (50% and 60% inhibition at 150 and 300 mM KC1, respectively), is not affected by 0.1-2 mM spermine, and exhibits a temperature dependence with an Arrhenius energy of activation, £a = 64.5 kJ, which is the same as that for tRNA aminoacylation with valine. Transfer RNA stimulates the reaction to a degree depending on the nature of amino acid, ATP concentration, pH and kind of buffer used, and KC1 and spermine concentrations. Changes of magnesium ion concentration in the range 0.25-10 mM do not affect the stimulation. The degree of stimulation by tRNA of the ATP pyrophosphohydrolase activity also does not depend on temperature. The tRNA apparently acts as an allosteric activator, which binds to the enzyme with £diss = 20 nM and increases KM for ATP from 9 to 100 µ . The ATM for amino acids is either not affected (ÁTM = 28 mM for alanine and 14 mM for serine) or slightly affected (£M = 9.6 mM for cysteine and 8.6 mM for threonine) by the presence of tRNA. Transfer RNA devoid of amino acid acceptance by periodate oxidation, albeit able to bind to the enzyme as well as intact tRNA, cannot produce these effects and does not inhibit the ATP pyrophosphohydrolase activity of valyl-tRNA synthetase. Lupin valyl-tRNA synthetase apparently rejects noncognate amino acids at the level of aminoacyl adenylate. The contribution of this pathway of rejection to the overall rejection of noncognate amino acids in the presence of tRNA is calculated to be 25%, 19%, 12%, 2.5% and 2% for cysteine, alanine, serine, threonine, and -aminobutyrate, respectively. e available estimates of error frequencies in protein biosynthesis (Loftfield, 1963;Loftfield & Vanderjagt, 1972;Edelmann & Gallant, 1977) indicate that the precision of

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Section: Resultsmentioning

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Valyl-tRNA synthetase from yellow lupin seeds: hydrolysis of the enzyme-bound noncognate aminoacyl adenylate as a possible mechanism of increasing specificity of the aminoacyl-tRNA synthetase

Jakubowski¹

1980

Biochemistry

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“…The enzyme blank contains everything except added tRNA. The aliquots were worked up as previously described (Loftfield and Eigner, 1967a). The 30-minute observations correspond to the incorporation of 0.35 m/j, mole of valine to 1 mg. of yeast tRNA and to 0.45 m^ mole of valine into 1 mg. of starfish tRNA, exactly the same saturating values as found with homologous enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…The aminoacylation of tRNA was carried out as previously described (Loftfield and Eigner, 1967a) and as summarized in the lengend of Figure 1.…”

Section: (D)mentioning

confidence: 99%

INTER-PHYLOGENETIC SPECIFICITY IN THE BONDING OF AMINO ACIDS TO tRNA

Loftfield¹,

Eigner²,

Nobel³

1968

The Biological Bulletin

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“…It seemed probable that raising the ionic strength would accelerate dissociation (Pingoud et al, 1973;Krauss et al, 1973;Loftfield and Eigner, 1967); therefore, we sought to determine, under Yarus and Berg's binding assay conditions, the effect of ionic strength on the overall reaction. In general under other conditions, it is known that higher ionic strengths slow aminoacylation reactions (Loftfield and Eigner, 1967;Taglang, et al, 1970;Smith, 1969; Holten and Jacobson, 1969;Yarus, 1972;Loftfield, 1972). The only reported exception to this generalization involves the tRNA and enzymes derived from halophilic bacteria (Griffiths and Bayley, 1969).…”

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confidence: 99%

The mechanism of the aminoacylation of transfer ribonucleic acid: enzyme-product dissociation is not rate limiting

Lövgren¹,

Pastuszyn²,

Loftfield³

1976

Biochemistry

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It has been proposed that the rate-limiting step in the synthesis of aminoacyl-tRNA is the rate at which the product dissociates from the enzyme. The experimental evidence supporting this hypothesis comes from work at low pH and low temperature (although the reaction has been argued to have the same mechanism under physiological conditions). We have reexamined the binding assay by which M. Yarus and P. Berg (1969) (J. Mol. Biol. 42, 171-189) measured the kd for dissociation of Enz-(Ile-tRNA). We find that when overall reaction and dissociation are measured under identical conditions the two rates are not the same. Moreover, while an increase in ionic strength greatly stimulates dissociation, the same increased ionic strength slows aminoacylation. Spermine accelerates overall aminoacylation without affecting dissociation. Because any change in a rate-limiting step must, by definition, cause a parallel change in the overall reaction, these observations prove that under these conditions the synthesis of Ile-tRNA is not limited by the rate of dissociation of Enz-(Ile-tRNA). Entirely similar observations were made for the dissociation of Enz-(Val-tRNA) and the overall synthesis of Val-tRNA at 0 degrees C, PH 5.0. In addition, valine enzyme isolated by nitrocellulose filtration during the course of an aminoacylation was shown not to be saturated with recently synthesized Val-tRNA. The enzyme was in equilibrium with uncharged substrate tRNA and with product Val-tRNA. E. W. Eldred and P. R. Schimmel ((1972) Biochemistry 11, 17-23) report that the formation of Ile-tRNA proceeds at two rates: (a) k = 2 X 10(-2)S(-1) until the enzyme is saturated with the first mole of product, and (b) k = 2 X 10(-3)S(-1) for subsequent cycles. We did not observe this behavior at any pH or temperature with four different amino acid:tRNA ligases. Because aminoacylation proceeds more rapidly than "dissociation" under some conditions, we believe that the binding assay measures not only enzyme-product dissociation but also other slower reactions such as aggregation or disaggregation of Enz-(AA-tRNA). In conjunction with recent studies from other laboratories, this work makes it unlikely that enzyme-product dissociation is the rate-limiting step in the synthesis of aminoacyl-tRNA either at low temperature and pH or under more nearly physiological conditions. From the effect of salt, it would appear that the rate of aminoacylation of tRNA is largely limited by the rate or extent of formation of Enz-(tRNA) (Loftfield, R. B., and Eigner, E. A. (1967), J. Biol. Chem. 242, 5355-5359). Using the binding assay of M. Yarus ((1972) Biochemistry 11, 2050-2060), we find the Kass for Enz-(Ile-tRNA) varies linearly with the Debye-Hückel function at ionic strengths of 0.1-0.4 from 10(8) to 10(6).

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Ionic Strength Effects in the Aminoacylation of Valine Transfer Ribonucleic Acid

Cited by 54 publications

References 8 publications

Valyl-tRNA synthetase from yellow lupin seeds: hydrolysis of the enzyme-bound noncognate aminoacyl adenylate as a possible mechanism of increasing specificity of the aminoacyl-tRNA synthetase

Valyl-tRNA synthetase from yellow lupin seeds: hydrolysis of the enzyme-bound noncognate aminoacyl adenylate as a possible mechanism of increasing specificity of the aminoacyl-tRNA synthetase

INTER-PHYLOGENETIC SPECIFICITY IN THE BONDING OF AMINO ACIDS TO tRNA

The mechanism of the aminoacylation of transfer ribonucleic acid: enzyme-product dissociation is not rate limiting

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