A rapid and convenient method for the detection and quantitative analysis of an amino acid:tRNA ligase·aminoacyl adenylate complex on DEAE-cellulose filter disks at pH 7.5 is presented. Results for L -phenylalanine: tRNA ligase (Escherichia coli K 10) demonstrate that 2 mol of L-phenylalanyl adenylate bind to 1 mol of enzyme. At 4°C, the decomposition of the ligase · aminoacyl adenylate complex was shown to follow first-order kinetics with a half-life of 16 min. With respect to the carboxylate-activating reaction catalyzed by L -phenylalanine:tRNA ligase the DEAE-cellulose matrix-bound enzyme appeared to be active.

The existence of two active siter per molecule of L-phenylalanine:tRNA ligase from Escherichia coli K(-10) has been demonstrated by isolation of the E-aminoacyl adenylate and tel filtration and the nitrocellulose filter assay at pH 5.0 revealed the same stoichiometry for the E-tRNAPhe comples as protection against degradation by snake venom phosphodiesterase and equilibrium gel filtration at pH 7.5. Using a fluorescence titration technique, it was found that the dissociation constant for ligase-tRNAPhe complex is decreased 20-fold when the hydrogen ion concentration is changed from pH 6.0 to pH 5.0. The existence of two active sites binding the aminoacyl adenylate intermediate was demonstrated by gel filtration and retention on DEAE-cellulose filters. "Burst" experiments indicated that two sites were involved in a rapid ATP consumption at conditions of catalytic amino acid activation. Furthermore, it was observed that the activated amino acid could be transferred from both sites to cognate tRNA.

l-Phenylalanine: tRNA ligase was inactivated and dissociated into subunits by exposure to guanidinium chloride, a pH of 2 or p-chloromercuribenzoate. After removal of the denaturant the enzyme quaternary structure was restored. In the case of acid denaturation it was shown that the specific activities of the restored and native enzyme were identical. At pH 2 and in guanidinium chloride dissociation proceeded directly to monomers while preliminary evidence was obtained that modification with p-chloromercuribenzoate at pH 7.5 was accompanied by dissociation into dimers. After exposure to pH 8.9 the modified dimers subsequently dissociated into monomers. The quaternary structures were differentiated by polyacrylamide gel electrophoresis, gel chromatography and kinetic analysis. Preparations of considerable purity were obtained for both monomers by polyacrylamide gel electrophoresis or sucrose gradient centrifugation; however, complete separation was accomplished only by gel chromatography of guanidinium-chloride-dissociated protein. Separated subunits had no detectable enzymic activity nor the ability to bind tRNAPhe and l-phenylalanine. However, when recombined, the enzymic properties were restored. An effect of the substrates and of magnesium was observed as a protection against the dissociation and inactivation of the enzyme when exposed to p-chloromercuribenzoate. An effect of magnesium was also observed during reassociation of the enzyme from pH-2-generated monomers. The rate of the reactivation was considerably enhanced by the presence of the cation, probably via a rapid formation of dimeric subunits. The results provide evidence that the integral quaternary structure of l-phenylalanine: tRNA ligase is a prerequisite for the functioning of the active site.

Formation of binary and ternary enzyme-ligand complexes was investigated for amino acid:tRNA ligases specific for L-isoleucine, L-leucine, and L-phenylalanine. Each of the enzymes exhibited synergistic binding when a substrate was substituted by a structurally related compound. The strength of coupling between the sites binding the amino acid and ATP was strongly dependent on the structure of ligands. The phenomenon was observed with the L-leucine and L-phenylalanine-specific enzymes only in the presence of magnesium. Spermine was inhibitory for L-phenylalanine:tRNA ligase. From the variation which structure of the strength of the observed synergism a correlation scheme was derived considering the ammonium group, the carboxylate group and the side chain of the amino acid, and the adenosine and triphosphate moieties of ATP. The strength of coupling between the subsites binding various combinations of these moieties was evaluated. We found that binding of the subgroups of the amino acid exerts an intramolecular synergism. The strength intramolecular synergism was similar to the strength of the intermolecular synergism observed for the simultaneous binding of an amino alcohol and ATP (or MgATP-2-). We have derived a molecular mechanism for the formation of the ternary enzyme-amino acid-ATP (or MgATP-2-) complex taking into account the synergistic phenomena. The complex is considered to involve electrostatic repulsion between the amino acid carboxylate and the ATP triphosphate moieties. When one of the negatively charged groups have been eliminated, the enzymatic rearrangement which facilitates the formation of this complex may be seen as a synergistic coupling.

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.

L-Phenylalanyl-tRNA synthetase has been reacted with N-bromoacetyl-[14C]Phe-tRNAPhe to yield covalently linked enzyme-N-acetyl-[14C]Phe-tRNAPhe. The labelled enzyme was dissociated in the presence of 4M guanidinium chloride and the subunits subsequently separated by gel chromatography. The elution pattern is indicative of covalent binding of the tRNA to the β-subunit of the enzyme.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEquilibrium analysis of L-Phe-tRNAPhe complexes with L-phenylalanyl transfer ribonucleic acid synthetase of Escherichia coli K 10Peter Bartmann, Till Hanke, Beate Hammer-Raber, and Eggehard HollerCite this: Biochemistry 1974, 13, 20, 4171–4175Publication Date (Print):September 1, 1974Publication History Published online1 May 2002Published inissue 1 September 1974https://pubs.acs.org/doi/10.1021/bi00717a016https://doi.org/10.1021/bi00717a016research-articleACS PublicationsRequest reuse permissionsArticle Views20Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

The molecular weight of l‐phenylalanyl‐tRNA synthetase was redetermined. A new value of 267 000 was obtained by high‐speed analytical ultracentrifugation and Sephadex G‐200 gel chromatography. The value is in contrast to the molecular weight of 180000 determined previouslyFrom the molecular weight 267000 and the sedimentation coefficient s20,w= 8.6 S the ratio f/f0= 1.67 was calculated. The magnitude of f/f0 is in accord with the molecular weight 183000 obtained from sucrose gradient centrifugation. The results indicate that the enzyme is highly hydrated and/or assumes the shape of a rod or a disc.The subunit composition was reinvestigated by sodium dodecylsulfate gel electrophoresis, guanidinium chloride gel filtration and by reactivation experiments of pH‐2 inactivated enzyme. The results are consistent with a subunit structure of the α2β2 type. Molecular weights of the subunits are 39000 and 94000, respectively.

L-Phenylalanyl-tRNA synthetase has been reacted with N-bromoacetyl-[ 14 C]Phe-tRNA Phe to yield covalently linked enzyme-N-acetyl-[ 14 C]Phe-tRNA Phe . The labelled enzyme was dissociated in the presence of 4M guanidinium chloride and the subunits subsequently separated by gel chromatography. The elution pattern is indicative of covalent binding of the tRNA to the β-subunit of the enzyme.

Binding of small ligands to the sites binding L-phenylalanine and ATP was measured by fluorescence titration technique. It is found that complex formation is not independent under conditions when both types of ligands are present in solution. The coupling is expressed as a synergistic binding rendering higher stabilities for complexes with ligand couples than expected on basis of separate binding of each ligand. In contrast, the substrate couple L-phenylalanine — ATP does not exhibit synergistic binding.

Concentration dependent association of the 17 S unit of horse spleen apoferritin leads to dimer formation at c ∼ 0.1 mg/ml; at c > 0.4 mg/ml trimers and higher aggregates are formed. Dissociation into subunits is not detectable at concentrations as low as 10−2 or 10−4 mg/ml using sedimentation velocity or gel chromatography, respectively. In accordance with iron incorporation into the preformed protein shell as the basic mechanism of ferritin formation, the results suggest the quaternary structure of apoferritin to be stable under quasi physiological conditions.