L-phenylalanyl-tRNA synthetase of E. coli. Active site specific binding of 2-p-toluidinylnaphthalene-6-sulfonate

Holler, Eggehard; Kosakowski, Heinz M.

doi:10.1016/0006-291x(73)91160-1

Cited by 10 publications

(5 citation statements)

References 11 publications

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“…(/) Effect of Ligand Binding on the Fluorescence of Enzyme-Bound TNS. In the presence of yeast phenylalanyl-tRNA synthetase, TNS exhibits a strong enhancement of fluorescence similarly to what has been described for the Escherichia coli isoleucyl-tRNA synthetase (Holler et al, 1971) and phenylalanyl-tRNA synthetase (Holler & Kosakowski, 1973). The addition of phenylalanine or ATP to a mixture containing enzyme and TNS leads to a decrease of the fluorescence intensity of the enzyme-bound TNS.…”

Section: Resultssupporting

confidence: 71%

Yeast phenylalanyl-tRNA synthetase: symmetric behavior of the enzyme during activation of phenylalanine as shown by a rapid kinetic investigation

Baltzinger¹,

Lin²,

Rémy³

1983

Biochemistry

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The adenylation of phenylalanine catalyzed by phenylalanyl-tRNA synthetase was investigated in the absence of tRNA, by rapid kinetic measurements using 6-(p-toluidinyl)naphthalene-2-sulfonate (TNS) as a nonspecific fluorescent reporter group. It is shown that each protomer of the enzyme is able to catalyze independently the adenylation of phenylalanine by ATP, as well as the reversion by pyrophosphate, at least in the absence of tRNA. The kinetic rate constants of synthesis and pyrophosphorolysis are respectively found equal to 100 +/- 20 s-1 and 150 +/- 50 s-1. The symmetric behavior of the enzyme is consistent with a symmetric binding of 2 mol of phenylalanine to the enzyme as shown by equilibrium dialysis experiments. The affinity of phenylalanyladenylate for the enzyme could be characterized by an equilibrium constant of 0.2 x 10(9) M-1.

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Section: Resultssupporting

confidence: 71%

Yeast phenylalanyl-tRNA synthetase: symmetric behavior of the enzyme during activation of phenylalanine as shown by a rapid kinetic investigation

Baltzinger¹,

Lin²,

Rémy³

1983

Biochemistry

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“…Contrary to what was observed for the Escherichia coli enzyme by Holler & Kosakowski (1973), the binding of TNS is not specific for the phenylalanine acceptor site of the yeast enzyme, as shown by Dibbelt (1981). An important fluorescence quenching is observed upon aminoacyladenylate formation, as already reported in the case of isoleucyl-tRNA synthetase by Holler & Calvin (1972).…”

Section: Methodsmentioning

confidence: 64%

Fast kinetic study of yeast phenylalanyl-tRNA synthetase: an efficient discrimination between tyrosine and phenylalanine at the level of the aminoacyladenylate-enzyme complex

Lin¹,

Baltzinger²,

Rémy³

1983

Biochemistry

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“…This approximation appears to be justified when reference is made to the similarity of values obtained from equilibrium measurements and the kinetic analysis of the ATP-[32P]PP¡ exchange (Mulivor and Rappaport, 1973). The assumption also finds support from the investigations with various substrate analogs (Kosakowski and Holler, 1973;Holler and Kosakowski, 1973;Holler et al, 1975). The dissociation constants used and the parameters evaluated from the slopes and intercepts on the basis of eq 1 are listed in Table II.…”

Section: Parameters Valuesmentioning

confidence: 79%

“…The values are similar in magnitude suggesting that the reporter group reflects binding of the amino acid to the high affinity site. We have previously reported that several molecules of the fluorescing compound interact with the enzyme, one of them exhibiting particular high affinity and enhanced fluorescence in the bound state (Holler and Kosakowski, 1973). Competition experiments with L-phenylalanine have demonstrated that dye and amino acid compete for the same site.…”

Section: Resultsmentioning

confidence: 99%

“…The accumulation predicts values for the Michaelis-Menten constants which are lower than those of the corresponding dissociation constants of ATP and L-phenylalanine. This idea has been treated in an earlier communication (Holler and Kosakowski, 1973). Since no other intermediate seems to be built up between the Michaelis-Menten complex of the amino acid activation reaction and the formation of the ternary enzyme-adenylate-pyrophosphate complex, the Michaelis-Menten constants of the ÁTP-[32P]PP¡ exchange reaction appear of similar magnitude as the dissociation constants (Holler et al, 1973, and references therein).…”

Section: Discussionmentioning

confidence: 99%

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L-Phenylalanine:tRNA ligase of Escherichia coli K10. Rapid kinetic investigation of the catalytic reaction

Bartmann¹,

Hanke²,

Holler³

1975

Biochemistry

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The kinetics of the amino acid activation and the transfer of the amino acid to tRNA have been investigated for L-phenylalanine:tRNA ligase of Escherichia coli K10 by stopped-flow and radioactive techniques. The rapid kinetics were followed by the observation of the displacement of the fluorescent dye, 6-p-toluidinylnaphthalene-2-sulfonate from the binding site of L-phenylalanine under conditions where a single active site of the enzyme was involved. The following results are of particular interest. (1) Equilibrium binding of L-phenylalanine and tRNAPhe indicates in each case two sites of interaction with an approximately tenfold difference of the binding affinity. (2) Experimental conditions of the kinetic investigation were chosen to favor reactions at the high affinity binding sites. Under those conditions, the rate constants have been evaluated at 1 mM magnesium to be in the range 12-25 sec-1 for the activation reaction and 42-77 sec-1 for the reverse, the variation of the values depending on those of the dissociation constants used for computation. The rate constant for the transfer reaction is 0.05 sec-1 and for the reverse 0.19 sec-1. The forward reaction is rate limiting for the overall reaction at single turnover and steady-state conditions. (3) All rate constants depend on the concentration of magnesium. Evidence is provided that the transfer occurs via a productive enzyme-tRNAPhe complex which is in a magnesium-dependent equilibrium with an unproductive complex, high magnesium favoring the former. The position of the tRNA-CCA end in the productive complex is such, that the fluorescent dye can be displaced by Phe-tRNAPhe. The thermodynamics of the overall reaction have been treated on the basis of the partial reactions. The free enthalpy of the completed reaction was calculated to be very close to zero. The significance of the adenylate intermediate is discussed with respect to the product inhibition expected on the basis of the tendency of tRNAPhe and L-phenylalanine to form tight complexes with the enzyme.

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L-phenylalanyl-tRNA synthetase of E. coli. Active site specific binding of 2-p-toluidinylnaphthalene-6-sulfonate

Cited by 10 publications

References 11 publications

Yeast phenylalanyl-tRNA synthetase: symmetric behavior of the enzyme during activation of phenylalanine as shown by a rapid kinetic investigation

Yeast phenylalanyl-tRNA synthetase: symmetric behavior of the enzyme during activation of phenylalanine as shown by a rapid kinetic investigation

Fast kinetic study of yeast phenylalanyl-tRNA synthetase: an efficient discrimination between tyrosine and phenylalanine at the level of the aminoacyladenylate-enzyme complex

L-Phenylalanine:tRNA ligase of Escherichia coli K10. Rapid kinetic investigation of the catalytic reaction

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