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E Holler

Publications and source records attributed to E Holler.

At least 181 records · Page 10Linked to original sources
PubMed1980

Kinetics of anticooperative binding of phenylalanyl-tRNAPhe and tRNAPhe to phenylalanyl-tRNA synthetase of Escherichia coli K10.

The kinetics of binding of Phe-tRNAPhe to phenylalanyl-tRNA synthetase were investigated by stopped-flow techniques employing 2-(p-toluidinyl)naphthalene-6-sulfonate as a reporter group. The kinetics were found to follow the concentration of Phe-tRNAPhe in terms of a saturation function. When added, tRNAPhe inhibited both the kinetics of association and the kinetics of dissociation. The kinetics of Phe-tRNAPhe binding have been analyzed to be a superposition of two reactions, the association to the phenylalanine-specific binding site and the association to the tRNA-specific binding site of the enzyme. The two modes are mutually exclusive. When tRNAPhe is added, the binding of Phe-tRNAPhe at the tRNA-specific site is suppressed by competitive inhibition. In binding to the tRNA-specific site, the unacylated tRNA affects the rates of association and dissociation of Phe-tRNAPhe binding at the Phe-specific site in terms of an antagonistic mechanism. The values of both rate constants are decreased depending on the degree of saturation of the enzyme with the unacylated tRNA before and after the association of Phe-tRNAPhe. A model is presented, and kinetic and equilibrium constants are determined.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Mechanism of phenylalanyl-tRNA synthetase of Escherichia coli K. 10. Modulation of catalytic properties by magnesium.

The association of phenylalanylptRNA and Mg2+ follows a biphasic concentration dependence as indicated by the active site directed fluorescent indicator 2-p-toluidinyl-naphthalene-6-sulfonate. The macroscopic dissociation constants are 0.16 +/- 0.03 and 4.1 +/- mM. The effect of Mg2+ on the association of enzyme and MgATP, on the synergistic binding of MgATP and L-phenylalaninol, and on the pre-steady-state synthesis and pyrophosphorolysis of the enzyme-phenylalanyladenylate complex in the absence and the presence of tRNA Phe has been measured by established equilibrium and stopped-flow techniques using 2-p-toluidinylnaphthalene-6-sulfonate. At 10 mM Mg2+, the association of enzyme and MgATP is biphasic with dissociation constants of 0.25 +/- 0.03 and 9.1 +/- 1.7 mM. At 2 mM Mg2+, a single dissociation constant of 5.0 +/- 0.5 mM is indicated. The coupling constant of the synergistic reaction is 15 at 1 mM Mg2+ and 290 at 10 mM Mg2+. The Hill constant of the sigmoidal dependence is 3.6. The strengthening of the synergism is believed to reflect a Mg2+-dependent coupling of the synergistic reactions at the two active sites of the enzyme, the coupling being negligible at 1 mM and maximal at 10 mM Mg2+. The pre-steady-state rate of adenylate synthesis is accelerated by the presence of Mg2+. The effect is to decrease the value of the Michaelis-Menten constant of MgATP. Another effect is to increase the rate constant when tRNA Phe is present. At subsaturating [MgATP], the [Mg2+] dependence of the observed rate constant is hyperbolical in the absence and sigmoidal (Hill constant, 3.5) in the presence of tRNA Phe. The rate of the pyrophosphorolysis is enhanced by a decrease of the Michaelis-Menten constant of MgPPi. The effects on the thermodynamics and kinetics parallel the occupancy of the low-affinity Mg2+-binding sites of the enzyme.

Adenosine Triphosphate↗
PubMed1979

Phenylalanyl-tRNA synthetase of Escherichia coli K 10. Multiple enzyme-aminoacyl-tRNA complexes as a consequence of substrate specificity.

The interaction between Phe-tRNA(Phe) or other acyl-tRNA derivatives thereof and phenylalanyl-tRNA synthetase of Escherichia coli K 10 has been investigated by nonequilibrium dialysis, by fluorescence titration in the presence of 2-p-toluidinylnaphthalene-6-sulfonate, by the kinetics of the aminoacylation of tRNA(Phe), and by the kinetics of the catalytic hydrolysis of Phe-tRNA(Phe). Phe-tRNA(Phe), or derivatives thereof, forms two types of complexes with the synthetase. One type involves the attachment of the phenylalanyl moiety to the phenylalanine-specific site of the enzyme, and the other type, to the tRNA(Phe)-specific binding site. They resemble alternative modes of a destabilized enzyme-product complex and are predicted on the basis of thermodynamic considerations. The two modes of binding of acyl-tRNA compete with each other. The attachment of Phe-tRNA(Phe) to the phenylalanine-specific site dominates. At equilibrium, this complex is present at a fourfold higher concentration than the other type of complex. The HNO2 deaminated Phe-tRNA(Phe) binds exclusively to the site specific for L-phenylalanine. On the contrary, Ile-tRNA(Phe) adds at 94.1% to the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) with this site leads to enzymatic hydrolysis into L-phenylalanine and tRNA(Phe). The complex involving the phenylalanine-specific site is hydrolytically unproductive. L-Phenylalanine acts as an activator of the hydrolysis by occupying the amino acid specific site and by shifting the equilibrium between the complexes toward the binding ot Phe-tRNA(Phe) at the tRNA(Phe)-specific site. The association of Phe-tRNA(Phe) at the phenylalanine-specific site does not interfere sterically with the binding of free tRNA(Phe). The sequential addition of free and aminoacylated tRNA(Phe) exhibits negative cooperativity. Such a mechanism could help to expel the product from the enzyme.

Amino Acyl-tRNA Synthetases↗
PubMed1977

Modification of L-isoleucyl-tRNA synthetase with L-isoleucyl-bromomethyl ketone. The effect of the catalytic steps.

The rapidly reacting cysteine-sulfhydryl group of L-isoleucyl-tRNA synthetase has been specifically alkylated with L-isoleucyl-bromomethyl ketone [Rainey, P., Holler, E. & Kula, M.-R. (1976) Eur. J. Biochem. 63, 419-426]. We have now investigated the catalytic and substrate binding properties of the modified protein by radioactive and fluorescence techniques. The rate constants for the transfer of AMP and isoleucine from the protein - adenylate complex to form ATP or Ile-tRNAIle were only 3% of those for native enzyme, whereas the rate constant for the formation of adenylate was essentially unchanged. The tendency to form synthetase - substrate complexes remained almost unchanged with the exception of L-isoleucine which exhibited a 20-fold reduction. Similarly, complex formation of L-isoleucinol together with its synergistic coupling to complex formation of ATP was partially inhibited. The results rule out the essential participation of the rapidly alkylatable cysteine-sulfhydryl group during catalysis.

Adenosine Triphosphate↗
PubMed1976

L-Phenylalanine: tRNA ligase of Escherichia coli K10. The effect of O replaced by S substitution on substrate and ligand binding properties of ATP.

The Km and V values of the tRNA-charging reaction have been measured for L-phenylalanine:tRNA ligase and the geometric isomers of adenosine 5'-O-(1-thio)triphosphate, adenosine 5'-O-(2-thio)triphosphate and for 5'-O-(3-thio)triphosphate. All ATP analogs were found to be substrates with values of Km similar or (up to 10-fold) higher, and with values of V in the range of 10--30% compared with the natural substrate. The dissociation constants of the binary enzyme-nucleotide and ternary enzyme-nucleotide-L-phenylalaninol complexes were analysed as a function of the position of the sulfur atom indicating those phosphate groups which are involved in an enzyme-triphosphate interaction. The results are consistent with a participation of the beta and gamma-phosphate in the binary complex formation and an additional interaction at the positions of the alpha and beta-phosphate groups in the ternary complexes.

Adenosine Triphosphate↗
PubMed1976

Labelling of L-isoleucine tRNA ligase from Escherichia coli with L-isoleucyl-bromomethyl ketone.

L-Isoleucine: tRNA ligase from Escherichia coli could be irreversibly inactivated by L-isoleucyl-bromomethyl ketone but not by L-isoleucyl-chromethyl ketone. The inactivation rate exhibited a saturation concentration dependence typical for an affinity reagent. L-Isoleucine provided 100% protection against inactivation at saturating concentration, whereas ATP, AMP, and pyrophosphate offered partial protection and tRNAIle, no protection. The ligase was labelled in preparative scale with L-[14C]isoleucyl-bromethyl ketone. The molar ration of label incorporated to enzyme inactivated was close to unity. The protein was subsequently subjected to tryptic digestion and the radioactive peptide isolated and identified. The labelled amino acid proved to be the same cysteine previously reported as being labelled with N-[14C]ethylmaleimide [Kula, M.-R. (1974) FEBS Lett. 46, 130-133].

Affinity Labels↗
PubMed1975

L-phenylalanine:tRNA ligase of Escherichia coli K10. A rapid kinetic investigation of the catalytic reaction.

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.

Adenosine Triphosphate↗
PubMed1975

Active site stoichiometry of L-phenylalanine: tRNA ligase from Escherichia coli K(-10).

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.

Adenosine Triphosphate↗
PubMed1975

Reaction of N-acetylglucosamine oligosaccharides with lysozyme. Temperature, pH, and solvent deuterium isotope effects; equilbrium, steady state, and pre-steady state measurements*.

Temperature jump and stopped flow methods were used to study at pH 7.0 the temperature dependence of elementary steps of the reactions of lysozyme with the beta(1 yields 4)-linked trimer, tetramer, and hexamer of N-acetylglucosamine. The steady state rate of cleavage of the hexasaccharide was determined as a function of temperature (5 degrees-40 degrees) and pH(2 to 8) in H-2O solution and as a function of pD(2.5 to 9.5) at 40 degrees in D-2O solution. The apparent enthalpies of the two ionizations of apparent pK 3.8 and 6.7 observed in measurements of k are 0 to 2 kcal/mol. The energy of activation determined for the pH optimum is 21.5 kcal/mol. The solvent deuterium isotope effect measured for k at the pH (pD) optimum is 1.5 And reflects isotope effects on pre-equilibrium steps and on the rate-determining step. Transfer from H-2O to D-2O solution produces 0.2 to 0.4 kcal/mol more negative free energies of saccharide binding and no changes in the enthalpies of binding. Pre-steady state, steady state, and equilibrium measurements indicate a pathway for the reaction of lysozyme with hexasaccharide. The results define for this mechanism the complete free energy profile and an essentially complete enthalpy profile. Three of the five observable ES complexes are present at nearly equal concentrations. The free energies of the transition states are within a range of 3 kcal. The enthalpies of productive enzyme-substrate complexes are about 5 kcal/mol greater than the enthalpies of nonproductive complexes. Changes in tryptophan fluorescence were observed for each elementary step, and changes in pK of Glu-35 for the isomerizations of nonproductive and productive complexes. The signal changes during formation of nonproductive complexes are the same for the oligosaccharides (ClcNAc)3 to (GlcNAc)6. The changes for productive complexes are similar but not identical with saccharides (GlcNAc)4 to (GlcNAc)6. Correlations of the present data with previous crystallographic and solution measurements indicate the structures of productive and nonproductive ES complexes and suggest that full interaction of the substrate with the enzyme active site is established in the rate-determining step.

Acetylglucosamine↗
PubMed1975

Productive and unproductive lysozyme-chitosaccharide complexes. Kinetic investigations.

Flow and relaxation methods were used to study the kinetics of oligosaccharides binding to lysozyme and the pre-steady-state kinetics of the lysozyme-catalyzed, hydrolysis of chitohexose. The minimal mechanism demonstrated, and the kinetics parameters pertaining to the elementary steps, allow interpretations of previous equilibrium and steady-state kinetic measurements which had yielded only complex constants, reflecting both productive and unproductive lysozyme-substrate complexes. In contrast to previous assumptions, the data presented in this paper provide evidence for "stable" productive lysozyme-substrate complexes. Our proposed mechanism utilizes structural information and accounts for the difference in efficiency of lysozyme-catalyzed hydrolysis of chitopentose and chitohexose.

Acetylglucosamine↗
PubMed1975

The catalytic mechanism of amino acid:tRNA ligases. Synergism and formation of the ternary enzyme-amino acid-ATP complex.

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.

Adenosine↗
PubMed1975

Productive and unproductive lysozyme-chitosaccharide complexes. Equilibrium measurements.

A method to determine both productive and unproductive lysozyme-chitosaccharide complexes has previously not been available. The method described in this paper uses a dye, Biebrich Scarlet, which forms a 1:1 complex with only part of the substrate binding site. Complex formation perturbs the spectrum of the compound and thus its dissociation constant can be determined (K-D equals 0.13 mM). The dissociation constants for three major enzyme-chitooligosaccharide complexes have also been determined: (1) chitooligosaccharides that bind only to sites A-C of lysozyme perturb the spectrum of the Biebrich Scarlet-lysozyme complex, without affecting the dissociation constant of the dye (K-u equals 0.01 mM); (2) chitooligosaccharides that interact with sites D-F displace the dye (K-S' equals 5-15 mM); (3) chitohexose forms a complex which involves the whole binding site and, therefore, also displaces Biebrich Scarlet. This complex, with a dissociation constant K-S equals 0.03 mM, is considered to be the productive one. The binding mechanism proposed on the basis of the results in this paper differs significantly from those considered previously.

Binding Sites↗
PubMed1975

Fluorescence and stopped-flow studies on the N in equilibrium F transition of serumalbumin.

In order to characterize the isomerization of serumalbumin in the acidic pH-range equilibrium and kinetic measurements of the intrinsic fluorescence of bovine serumalbumin and human serumalbumin were performed. Additional experiments with modified bovine serumalbumin made use of substituted 1.9 benzoxanthene dyes as SH-specific extrinsic fluorophores. The intrinsic fluorescence (lambda exc = 275 nm) shows a pH-dependent shift of the maximum of fluorescence emissions which correlates with the N in equilibrium F isomerization. This and the acid expansion at pH less than 3.5 is indicated by the pH-dependence of the fluorescence intensity at 350 nm. While tyrosine fluorescence is increased in all steps of the transition, tryptophane fluorescence is decreased in a different way for BSA (2 trp/molecule) and HSA (1 trp/molecule), the latter showing the N in equilibrium F transition only. Combining the tryptophan fluorescence data with the results from the SH-specific modification of BSA the conclusion may be drawn that the tryptophan residues in BSA and the SH-group belong to different domains of the molecule. Stopped-flow experiments prove the N in equilibrium F' and the F' in equilibrium F transitions to be separable along the time axis, the relaxation times being in the range between 40-50 and 300-600 msec respectively. For the "expansion" the kinetic constants critically depend on the initial pH conditions of the solutions. The backward reaction F leads to N seems to be a multistep isomerization process which is characterized by relaxation times greater than 1 sec.

Animals↗